The high mortality rate in nursing homes during the COVID-19 pandemic may be linked to psychological disorders in staff. Hence, we assessed the prevalence and associated factors of probable post-traumatic stress disorder (PTSD), anxiety, depression, and burnout of nursing home staff during the COVID-19 pandemic in a cross-sectional study including 66 randomly selected nursing homes in southern France. 537 of the contacted 3 821 nursing home workers (14.0%) responded between April and October 2021. We collected information on center organization, severity of COVID-19 exposure, and socio-demographic information in an online survey. The prevalence of probable PTSD (PCL-5), anxiety and depressive disorders  and the sub-scores of burnout syndrome  were assessed. Probable PTSD was reported in 115/537 responders (21.4% (95% CI [18.0%–24.9%])). After adjustment, low-level exposure to COVID-19 in nursing home residents , fear of managing COVID-19 residents , conflicts with residents , conflicts with colleagues , cancellation of leave  and temporary worker employment  were associated with higher prevalence of probable PTSD. The prevalence of probable anxiety and depression were 28.8% (95% CI [24.9%–32.7%]) and 10.4% (95% CI [7.8%–13.1%]), respectively. Psychological disorders were observed in nearly one third of nursing home workers during the COVID-19 pandemic. Hence, continuous surveys and preventive measures are needed in this particularly at-risk population. Since December 2019, the coronavirus SARS-CoV-2 has caused a worldwide outbreak of respiratory illness COVID-19, ranging from asymptomatic to mild and severe progressive pneumonia, leading to death with an overall lethality of around 3% .By November 2022, the COVID-19 pandemic had caused near 160 000 deaths in France . Older pIn the general population, quarantine, lockdown and isolation contributed to Post-Traumatic Stress Disorder (PTSD) syndrome in about 30% of the overall population . AnotherNursing homes workers are at particular risk of developing psychological consequences such as PTSD, anxiety disorders, depression, and burnout syndrome (BOS) according to studies performed in the first year of COVID-19 pandemic . AnotherSuch psychological consequences are likely to be due to: (1) the management of COVID-19 residents, with potential mortality; (2) the risk of infecting oneself or others; (3) conflicts with residents’ relatives, for instance as a consequence of visit restriction policies or following infection of residents ; (4) deaGiven the significant impact with long-term consequences for nursing home workers of the COVID-19 pandemic, studies evaluating psychological distress in the later phases of the pandemic are important in this population. Furthermore, research involving greater samples may help to identify specific factors related to probable psychiatric disorders in this population. This study aimed to assess the prevalence and associated factors of probable PTSD in workers of nursing homes in the region Occitanie, in Southern France. We also assessed signs of probable anxiety and depression, BOS and their related factors.n = 817) of the region were informed of the study and 100 nursing homes were randomly selected, according to department and COVID-19 status. Centers were classified into three categories according to their COVID-19 status listed by Santé Publique France database at study initiation: Institutions with at least one COVID-19 resident (COVID + ), with no COVID-19 resident (FREE-COVID), and unknown status (UNK). Nursing homes which refused to participate were replaced at random by one with the same COVID-19 status in the same department.A multicenter cross-sectional nursing home-based online survey was carried out among nursing home workers in Occitanie, a region of 6 million inhabitants subdivided into 13 departments, in Southern France. This study was approved by the national ethics committee , been carried out in accordance with the Declaration of Helsinki and registered on clinicaltrials.gov (NCT04916275) . A free Different categories of the staff;Number of beds in 2020;Number of residents in 2019 and 2020 (who stayed at least one day each year);Type of institution ;Adaptation of the nursing homes during the COVID-19 pandemic ;Number of residents infected by COVID-19 and number of deaths among COVID-19 residents.The center questionnaire focused on the organization of the nursing home:Socio-demographic data during the pandemic;Professional characteristics , experience during the COVID-19 pandemic ;Three validated questionnaires to screen for probable psychiatric disorders: the PTSD Checklist for DSM-5 (PCL-5)  to evaluThe personnel self-questionnaire collected:The survey included two questionnaires:All staff of participating nursing homes, caregivers and non-caregivers, were eligible to participate. The inclusion criteria were: aged 18 and older, fluent French, and access to the internet. For completing the personal questionnaire, a site-specific web link and QR code were provided to members via the nursing home local investigator. The recruitment was initially planned from May to June 2021.The primary outcome was the presence of probable PTSD defined by PCL-5 score ≥32 .The secondary outcomes were the presence of probable anxiety or depression, and BOS. Anxiety and depression were separately screened with HADS questionnaire for total score between 0 and 21, where a score of 0 to 7 indicated absence of disorder; 8 to 10 a doubtful disorder; and 11 to 21 a probable disorder . BOS wasCOVID-19 exposure used the classification used by the French Agency “Direction de la Recherche des Études de l’Évaluation et des Statistiques” (DREES) in four categories: COVID-19 critical exposure: ≥ 10% or 10 COVID-19 resident deaths; COVID-19 severe exposure: ≥ 33% or 30 COVID-19 residents; unqualified episode: at least one COVID-19 resident; FREE-COVID: no resident with COVID-19 . To consNursing home data included: legal status; number and categories of worker; recruitment of temporary workers during the pandemic; and types of preventive measures .Individual data included responder characteristics: age, sex, changes in life-style during the pandemic ; and professional characteristics .All these factors were analyzed as potentially associated with probable PTSD, anxiety, depression, and BOS.Questionnaires were tested by 20 independent caregivers not involved in nursing home care before the start of the study. Moreover these questionnaires could be completed from any web or mobile device, via the anonymous REDCap link .Assuming a baseline prevalence of PTSD of 25% (FREE-COVID group) and an absolute increase of 10% in the COVID group, i.e. 35%, respecting the imbalance of the groups from the preliminary data (ratio COVID/FREE-COVID = 2.45), with a two-sided alpha risk of 5% and a power of 90%, the number of participants needed is 1075 , 27. To PTSD was classified as “probable” (PCL-5 score ≥ 32) versus no PTSD (PCL-5 score < 32) and presented with 95% confidence interval (95% CI) . The prep-value ≤ 0.20 in the univariate analysis were selected for multivariate analysis. To account for the hierarchical nature of the data , the nursing home was defined as a random factor and included in a multilevel logistic regression with backward selection strategy at the 5% threshold. As main exposure, nursing-home COVID status was forced in the model. Adjusted odds ratio (AOR) were provided with 95% CI.PTSD associated factors were analyzed in a univariate analysis. The qualitative variables (expressed as numbers and percentages) were compared according to the PTSD by the chi-square test or the Fisher exact test as appropriate. The links between the explanatory variables and PTSD variable were expressed by odds ratios and 95% CI by the Wald method. Covariates with a The same analysis strategy was applied to evaluate the prevalence of anxiety and depression and their associated factors. A multilevel polytomous logistic regression with proportional-odds cumulative logit model was used to search for factors associated with anxiety and depression classified in a three-level ordinal variable. The scores of the emotional exhaustion, depersonalization and personal accomplishment subscales were expressed as mean, standard deviation, median and interquartile range (IQR). The associated factors to the three sub-scores were assessed with a multilevel multiple linear regression model. The same variable selection strategy was used as for the previous models. Pearson correlation coefficients between PTSD, anxiety, depression, emotional exhaustion, depersonalization and personal accomplishment scores are provided with their 95% CI. All statistical analyses used SAS statistical software, version 9.4 (SAS Institute Inc).Of the 817 eligible nursing homes, 134 were invited to participate and 66 agreed to respond , anxiety (n = 521) and depression (n = 527) and burnout (n = 510)  with no difference between responders from FREE-COVID  and COVID+  nursing homes.The multivariate analysis identified five factors associated with greater frequency of probable PTSD , depression  and emotional exhaustion  scores  with no difference between responders from FREE-COVID  and COVID+  nursing homes. According to the multivariate analysis, fear of managing COVID-19 residents, conflicts with residents, age between 30 and 49 year-old, family environment alteration and distrust or low confidence in the data relayed by the media, were associated with anxiety disorder (Table 2.7%) witA probable depressive disorder was reported in 55/527 responders (10.4% 95% CI [7.8%–13.1%]) with no difference between responders from FREE-COVID  and COVID+  nursing homes.According to the multivariate analysis, comorbidity at risk of severe COVID-19, distrust or low confidence in media communication about personal preventive measures, conflicts with residents and colleagues, negative reaction to canceled leave and insufficient information provided by the institution, were associated with depressive disorder . According to the multivariate analysis, fear of managing COVID-19 residents, conflicts with residents and colleagues, distrust or low confidence in the data relayed by the media, negative reaction to canceled leave and personnel shortage were independently associated with a greater emotional exhaustion. In contrast, the perception of excellent information from the institution decreased emotional exhaustion . According to the multivariate analysis, fear of managing COVID-19 residents, conflicts with residents and their relatives, distrust or low confidence in media communication about personal preventive measures, negative reaction to canceled leave and insufficient level of personal protective equipment were associated with a greater depersonalization. Nursing homes that used more temporary workers during the pandemic than before were associated with a greater depersonalization. Having transferred COVID-19 residents to another facility decreased depersonalization . According to the multivariate analysis, fear of managing COVID-19 residents, conflicts with residents’ relatives and being a non-healthcare worker were associated with a lower personal accomplishment, while seeking information with the same frequency or more often during the pandemic than before was associated with greater personal accomplishment . Depersonalization was negatively but less correlated with personal accomplishment  and quarantining. Indeed, PTSD, anxiety, depression and burnout have been widely reported among the general population and in healthcare workers  and may The restrictive visiting policies implemented in nursing homes exposed residents and employees (caregiver and administrative agents) to psychological disturbances . In a quA cross-sectional survey of 390 nursing home staff performed during the third wave of COVID-19 pandemic in Ireland (from 20 November 2020 to 4 January 2021) found PTSD in 45.1% of all staff, low mood , and suicidal behavior (13.8% with no difference between job categories) . InteresThis study highlighted several factors associated with PTSD, anxiety, depression, and symptoms of BOS. The implication of conflicts with patients  and the Although the fear of managing COVID-19 resident was associated with a higher prevalence of PTSD, a high level of exposure to COVID-19 in nursing home residents was associated with a lower one. This surprising finding could be explained by the degree of information and expertise in workers managing COVID-19 patients.We must acknowledge certain limitations. First, participation rate was around 10%, as classically reported in cross-sectional survey without possibility of personal reminder (respecting responder anonymity). The personnel participation varied between institutions (1–83%). Only 66 of 134 contacted nursing homes agreed to participate. The timing of our study  was perhaps too long after the start of the pandemic with participant weariness leading to a low response rate. Second, due to its cross-sectional survey design, our study could only determine associated factors with probable PTSD, anxiety, depression, and BOS, without any causal relationship. To isolate risk factors of these psychological disorders, cohort or case control designs might have been more appropriate, but none have been performed so far. Third, data on participant follow-up by mental health professionals and the intake of psychotropic medication at the time of the survey was not included in the analysis. Hence, prevalence of psychological disorder may have been underestimated. Yet, these factors were also not taken into account in other similar studies conducted in nursing homes . Fourth,This study shows that the COVID-19 pandemic was accompanied by a risk of developing psychological disorders in nearly a third of nursing home workers. Some factors linked to the COVID-19 pandemic (fear of managing COVID-19 patient), and others such as conflicts with residents and colleagues, and cancellation of leave were associated with a higher prevalence of probable PTSD, anxiety, depression and BOS. These findings could plea for continuing surveying nursing home workers, as this population is particularly at risk for psychological disorders. These surveys could lead to preventive and therapeutic measures.Supplemental material (Tables)Supplemental material


National and regional carbapenemase-producing Enterobacterales (CPE) surveillance is essential to understand the burden of antimicrobial resistance, elucidate outbreaks, and develop infection-control or antimicrobial-treatment recommendations.This study aimed to describe CPE and their epidemiology in Norway from 2015 to 2021.A nationwide, population-based observational study of all verified clinical and carriage CPE isolates submitted to the national reference laboratory was conducted. Isolates were characterised by antimicrobial susceptibility testing, whole genome sequencing (WGS) and basic metadata. Annual CPE incidences were also estimated.Escherichia coli and Klebsiella pneumoniae, including high-risk clones also detected globally. Most CPE isolates were travel-related . Although local outbreaks and healthcare-associated transmission occurred, no interregional spread was detected. Nevertheless, 18% (70/389) of isolates not directly related to import points towards potentially unidentified transmission routes. A decline in travel-associated cases was observed during the COVID-19 pandemic.A total of 389 CPE isolates were identified from 332 patients of 63 years median age (range: 0–98). These corresponded to 341 cases, 184 (54%) being male. Between 2015 and 2021, the annual incidence of CPE cases increased from 0.6 to 1.1 per 100,000 person-years. For CPE-isolates with available data on colonisation/infection, 58% (226/389) were associated with colonisation and 38% (149/389) with clinical infections. WGS revealed a predominance of OXA-48-like  and NDM  carbapenemases in a diversified population of The close-to-doubling of CPE case incidence between 2015 and 2021 was associated with foreign travel and genomic diversity. To limit further transmission and outbreaks, continued screening and monitoring is essential. What did you want to address in this study?Carbapenems are effective antibiotics used to treat serious and multidrug-resistant bacterial infections. Carbapenemase-producing Enterobacterales (CPE) are bacteria that can cause difficult to cure infections and challenge the usefulness of carbapenems. Besides the ability to infect people, CPE can also colonise individuals. We wanted to describe CPE in Norway, their epidemiology in 2015–2021 and characteristics of patients with CPE.What have we learnt from this study?During the study, 332 patients with CPE were identified. Analyses found ca 58% of patients colonised. Between 2015 and 2021, CPE annual incidence in Norway nearly doubled, partly via travel, with people possibly importing diverse CPE, and partly via local-level CPE transmission between patients in healthcare. For ca 18% of patients with CPE, CPE acquisition could not be linked to import and CPE transmission routes remained unclear. What are the implications of your findings for public health?The increase of CPE incidence during the study period and the large proportion of CPE identified as colonisation underline that continued vigilance for CPE in Norway is important. Risk assessments and maintained emphasis on screening, surveillance and infection control are needed to limit the further spread of these bacteria, as well as their transmission in hospitals, and their establishment in both the healthcare setting and the community.Escherichia coli and Klebsiella pneumoniae associated with significant morbidity and mortality . Th. Th17]. nd Italy . The relWe have previously reported that the CPE epidemiology in Norway is characterised by sporadic cases . AlthougIn 2007–2014 only 15% of CPE isolates were identified through screening . The larE. coli/K. pneumoniae are the dominant combinations observed in Asia and Europe [E. coli ST38 and OXA-48-producing K. pneumoniae ST392) or emergence of successful carbapenemase-variants such as NDM-5 in a diversity of genetic backgrounds including global high-risk ExPEC and K. pneumoniae clones [The high proportion of CPE isolates associated with import  and the large diversity of species, carbapenemases and STs likely illustrate the global epidemiology of CPE. OXA-48-like and/or NDM-producing d Europe ,41 and 9e clones ,35. GenoThe emergence of OXA-244 in Norway and the high proportion of OXA-48 variants also challenge current diagnostic screening algorithms in CPE detection . In our The study has some limitations, such as the lack of antimicrobial susceptibility data on new antimicrobial agents such as cefiderocol, and the β-lactam-β-lactamase inhibitor combinations meropenem–vaborbactam and imipenem–relebactam. In addition, the susceptibility data were acquired by a broth microdilution method that deviates slightly from the International Organization for Standardization (ISO) standard . MoreoveA strength of this study, however, is the national requirement of mandatory reporting of CPE-cases including patient characteristics and submission of suspected CPE-isolates to a reference laboratory from all clinical microbiological laboratories. This ensures national population-based data of confirmed CPE-cases. Nevertheless, the observed incidences should be interpreted with caution as there are relatively few CPE cases.The near doubling of CPE-case incidence rates in Norway is associated with multiple factors including travel abroad and import of epidemiological successful high-risk clones and carbapenemase-variants. The relative high proportion of cases  not directly related to import underlines the potential importance of unidentified transmission routes. The single hospital and intraregional outbreaks are of concern. The downward incidence rate and travel-associated cases during the COVID-19 pandemic is likely to be reversed as international travel increases. Generic infection control measures and adherence to rectal screening recommendations as well as close monitoring of the molecular CPE epidemiology is of particular importance to avoid further transmission, outbreaks and establishment of CPE in Norway.


Peripheral blood cytopenia, a frequent presenting symptom in pediatric patients, can be caused by bone marrow failure (BMF). Timely identification of patients with non-reversible BMF is of crucial importance to reduce the risks of invasive infections and bleeding complications. Most pediatric patients with severe persistent cytopenia, independent of the underlying cause, are offered allogeneic hematopoietic stem cell transplantation (HSCT) as curative therapy. Here we report on our management guidelines and HSCT outcomes of pediatric BMF patients to pinpoint improvements and future challenges. We formulated recommendations based on this 50 years’ experience, which were implemented at our center in 2017. By analysis of the HSCT cohort of 2017–2023, the 5-year outcome data is presented and compared to historical outcome data. In addition, outcomes of patients transplanted for identified inherited bone marrow failure syndromes (IBMFS) are compared to severe aplastic anemia (SAA) outcomes to underline the often multiorgan disease in IBMFS with implications for long-term survival. Survival of pediatric patients with irreversible BMF has improved tremendously. SAA patients transplanted after 2017 had a superior 5-year overall (OS) and event-free survival (EFS) of 97% and 85% compared to 68% and 59% in the cohort transplanted before 2017 (p = 0.0011 and p = 0.017). A similar trend was seen for BMF, with an OS and EFS of 89% for those transplanted after 2017 compared to 62% and 59% (p > 0.05). This improvement is mainly related to better survival in the first months after HSCT. The long-term survival after HSCT is lower in IBMFS patients as compared to SAA patients due to secondary malignancies and multiorgan toxicity.Conclusion: Unbiased protocolized in-depth diagnostic strategies are crucial to increase the frequency of identifiable causes within the heterogeneous group of pediatric BMF. A comprehensive approach to identify the cause of BMF can prevent treatment delay and be useful to tailor treatment and follow-up protocols.   The online version contains supplementary material available at 10.1007/s00431-023-05093-y. Non-reversible bone marrow failure (BMF) in pediatric patients is caused by a broad spectrum of underlying diseases, including inherited bone marrow failure syndromes (IBMFS), (pre)malignant disease, and (idiopathic) aplastic anemia (AA) –3. In upTimely recognition of (irreversible) BMF and identification of the underlying cause of BMF result in reduced risks of invasive infections and bleeding complications. This requires guidelines for a consistent diagnostic approach for pediatric patients suspected of BMF. In these guidelines, in addition to morphological and histological examination of bone marrow aspirates and biopsies, molecular techniques and functional assays are of increasing value. In general, genetic screening is initiated in case of extra-hematological physical abnormalities and/or a positive family history , 13. HowPediatric BMF patients are transplanted in our center for over 50 years. The first severe AA (SAA) patient (BMF of unknown origin at the time) was transplanted in 1971 and the first patient with severe BMF due to FA was transplanted in 1972. During the years following, conditioning regimens developed from toxic myeloablative to more reduced intensity and toxicity regimes . This database includes the clinical data of all pediatric patients who received an HSCT in the LUMC as of 1965. MDS or malignant disorders causing BMF, other than MDS-RCC were not included in the analysis. Between 1971 and 2022 a total of 265 transplantations were performed in 226 pediatric patients with BMF , event-free survival (EFS) and GVHD. EFS or OS was defined as date from initial diagnose to date of relapse, second HSCT and/or death by any cause. Patient characteristics include age and gender. Donor characteristics cover donor type  including both matched and mismatched donors) and source  or umbilical cord blood (UCB). Transplantation characteristics include conditioning regimen, GVHD prophylaxis and the number and date of transplantation. Missing data were collected from the electronical patient records by the treating physician.Statistical analysis was performed by using RStudio (R4.2.2). Characteristics of the patients and donors were described. For analyzing continuous variables among the cohorts, the Wilcoxon rank sum test was used. Pearson’s Chi-squared test was used for categorical data. For the survival analyses, Kaplan-Meier curves were plotted including 95% confidence intervals. Upon last date of follow-up, patients without an event were censored. The 5-year OS and EFS were compared between the cohorts transplanted before and after 2017 using a Log-rank test. In addition, long-term (30-year) OS and EFS of patients transplanted before 2017 was visualized. 5-year OS per donor type was compared using Kaplan-Meier curves and a Long-rank test. Median follow-up time was computed using the reversed Kaplan-Meier method. Endpoint of this study is 01-01-2023.The clinical characteristics of the patients included are summarized in Table SAA patients transplanted after 2017 had a superior 5-year OS and EFS, respectively 97% and 85% as compared to 68% and 59% in the cohort transplanted before 2017  and chronic GVHD incidences were low  and hypoplastic bone marrow without increased risk of malignant transformation (such as in SAA) –17. AlsoAlso, the treatment of pediatric BMF patients is accompanied with challenges. The majority of these patients present with or develop severe cytopenia or risk indicators for hematologic malignancies. Thereby for most patients, HSCT is an attractive and often the only curative treatment option. Based on our long-term experience in HSCT for pediatric patients with BMF as presented here, we draw the following conclusions. HSCT has an unequaled curative potential to restore BM defects independent of the underlying cause. Moreover, with the current successes of HLA-mismatched transplantations by effective GVHD prevention such as PT-Cy or alpha/beta T-cell depletion, and reduced toxicity conditioning regimens, HSCT is increasingly offered as the first choice of treatment to pediatric BMF patients. However, short- and long-term (event-free) survival rates are evidently dependent on the management of the patients before and after transplantation. A structured multidisciplinary approach directed towards unbiased but swift diagnostics at the suspicion to diagnosis stage together with maximal supportive care to prevent infections and bleeding result into significant gain of survival rates within the first 3 months after the onset of the disease. This is underlined by the significant improved overall and disease-free survival rates of BMF patients transplanted after 2017 independent of the underlying cause. In addition, especially in patients with IBMFS, protocolized approaches to systematically screen for late effects post HSCT are of crucial importance. The second decline in survival of these patients 10 to 20 years after HSCT, often explained by secondary malignancies, lung- and liver fibrosis and other organ failures, underlines the essence of personalized follow-up protocols. This wide variety of long-term toxicities including chronic GVHD, delayed or unbalanced immune reconstitution, iron overload, developmental delay and psychosocial impairment and malignant disease require a multidisciplinary approach guided by centers of expertise .Supplementary file1 (DOCX 21 KB)Below is the link to the electronic supplementary material.


Solid tumors account for about 30% of all pediatric cancers. The diagnosis is typically based on histological and molecular analysis of a primary tumor biopsy. Liquid biopsies carry several advantages over conventional tissue biopsy. However, their use for genomic analysis and response monitoring of pediatric solid tumors is still in experimental stages and mostly performed retrospectively without direct impact on patient management. In this case series we discuss six clinical cases of children with a solid tumor for whom a liquid biopsy assay was performed and demonstrate the potential of liquid biopsy for future clinical decision making.We performed quantitative real-time PCR (RT-qPCR), droplet digital PCR (ddPCR) or reduced representation bisulphite sequencing of cell-free DNA (cfRRBS) on liquid biopsies collected from six pediatric patients with a solid tumor treated between 2017 and 2023 at the Princess Máxima Center for Pediatric Oncology in the Netherlands. Results were used to aid in clinical decision making by contribution to establish a diagnosis, by prognostication and response to therapy monitoring.In three patients cfRRBS helped to establish the diagnosis of a rhabdomyosarcoma, an Ewing sarcoma and a neuroblastoma (case 1-3). In two patients, liquid biopsies were used for prognostication, by MYCN ddPCR in a patient with neuroblastoma and by RT-qPCR testing rhabdomyosarcoma-specific mRNA in bone marrow of a patient with a rhabdomyosarcoma (case 4 and 5). In case 6, mRNA testing demonstrated disease progression and assisted clinical decision making.This case series illustrates the value of liquid biopsy. We further demonstrate and recommend the use of liquid biopsies to be used in conjunction with conventional methods for the determination of metastatic status, prognostication and monitoring of treatment response in patients with pediatric solid tumors. Solid tumors account for about 30% of all pediatric cancers. The most common extra-cranial solid tumors in children are neuroblastoma, rhabdomyosarcoma, Wilms tumor and bone sarcomas . In geneLiquid biopsies allow detailed analysis of the tumor profile in body fluids. Circulating sources of genomic information, such as cell-free tumor DNA (ctDNA), circulating tumor cells and tumor-derived mRNA, can reveal characteristics that are relevant for clinical decision making, but can be absent in a tumor biopsy –19. LiquCurrently, the use of liquid biopsies for genomic analysis and monitoring of pediatric solid tumors is still in experimental stages and mostly performed retrospectively without direct impact on patient management. However, incidental cases do demonstrate their potential for future clinical decision making. Many techniques that are classically used for tissue analysis can also be applied to liquid biopsies, including various sequencing approaches for cell-free DNA (cfDNA), tumor-specific PCR  and digital PCRs) and methylation analysis Figure 1In this report, we describe the clinical application of RT-qPCR, droplet digital PCR (ddPCR) and methylation analysis through reduced representation bisulphite sequencing of cell-free DNA (cfRRBS). RT-qPCR has been shown to be a sensitive technique for the detection and monitoring of MRD in several pediatric solid tumors –27. Our MYCN is an indicator for high-risk disease . RASSF1A is the most frequently inactivated tumor suppressor gene in cancer, most often inactivated by hypermethylation of the promotor  at -80°CC. PB samples were collected and processed the same way and stored in in PAXgene blood RNA tubes  at -20°CC, or in Trizol  at -80°CC. DNA and RNA isolation and cDNA synthesis was performed as previously described , 32, 40.PHOX2B, TH and CHRNA3, and GAP43 and DBH for BM and PB  aspirates and/or peripheral blood (PB). For neuroblastoma, a multiplex (MPX) adrenergic marker panel was used which includes M and PB , 29. Ford MEGF10 . Expressk et al. –30 To caRASSF1A-M in ctDNA was performed using the method described by Van Zogchel et al.  primer-probe set were added in a duplex PCR reaction. MYCN copy number was then determined by calculating the ratio of MYCN to the reference gene  was performed as previously described . The diaSee For a summary of the conventional diagnostics and clinical value of liquid biopsy for each case, see A 3-year-old girl presented with lethargy, abdominal pain, vomiting and a palpable abdominal mass Table 1.123I]mIBG imaging  were detected. Because of the heterogeneous [123I]mIBG uptake, an [18F]FDG-PET CT was performed  and vanillylmandelic acid (VMA) and slightly elevated levels of dopamine, 3-methoxytyramine (3MT) and norepinephrine. Histologically, the primary tumor biopsy did not express common neuroblastoma markers . SNP-array showed a gain of chromosome 17q and next-generation sequencing detected a SMARCA4 mutation . Based on DNA-methylation , the tumor was classified as a rhabdoid tumor (score 0.73). Methylation analysis of cfDNA from the blood plasma was performed to aid in the diagnosis of this case. Deconvolution of cfRRBS data estimated both rhabdoid tumor (44.2% tumor fraction) and neuroblastoma (22.8% tumor fraction). In addition, RT-qPCR was performed on diagnostic BM and PB samples using the neuroblastoma MPX-panel, which came back positive for mRNA markers CHRNA3 and GAP43  calcified primary tumor lesion remained , or circulating tumor cells (CTCs). However, bone marrow, cerebrospinal fluid, urine and even saliva or pleural effusions may also be sampled. Novel components for analysis include extracellular vesicles, (tumor-educated) platelets, proteins and immune cells , 23. In Molecular profiling of tumor-derived cfDNA strongly correlates with tumor tissue , 38, 57.Sample collection is mostly minimally invasive and can be repeated throughout treatment and follow-up, providing new opportunities to monitor disease burden and clonal evolution , 61, 62.BRAF, EGFR, or KRAS, do not translate to the pediatric population where such recurrent mutations are rare. Additionally, while sample volume is not typically a concern in adults, pediatric assays must be optimized to accommodate their smaller circulating volume. Together, these discrepancies underline the need to develop pediatric assays independently  cL plasma .Liquid biopsy shows great potential to assist with diagnosis, prognosis and disease monitoring in pediatric solid tumors. As liquid biopsies in pediatric oncology have not yet moved from bench to bedside, many techniques still await standardization. However, in a first step towards translation and clinical implementation, the techniques described in this case series are now being validated in international prospective trials. RT-qPCR and ddPCR will be validated for neuroblastoma in the ongoing SIOPEN HR-NBL2 trial. cfRRBS has been included in the ongoing SIOP RTSG-UMBRELLA trial for renal tumors. For rhabdomyosarcoma, the RNA panel and cfDNA analysis will be validated in the FaR-RMS trial. We recommend that liquid biopsies be used in conjunction with other conventional methods for the determination of tumor dissemination, prognostication and the monitoring of treatment response for pediatric patients with solid tumors. However, as illustrated by the case series in this paper, liquid biopsies may also be of crucial importance in cases where conventional techniques fall short.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.Written informed consent was obtained from the minor(s)’ legal guardian/next of kin for the publication of any potentially identifiable images or data included in this article.NG and AB are shared first author. All authors contributed to the article and approved the submitted version.


Unhealthy lifestyle behaviours such as smoking, high alcohol consumption, poor diet or low physical activity are associated with morbidity and premature mortality. Public health guidelines provide recommendations for adherence to these four factors, however, their impact on the health of older people is less certain.The study involved 11,340 Australian participants (median age 7.39 ) from the ASPirin in Reducing Events in the Elderly study, followed for a median of 6.8 years . We investigated whether a point-based lifestyle score based on adherence to guidelines for a healthy diet, physical activity, non-smoking and moderate alcohol consumption was associated with all-cause and cause-specific mortality.In multivariable adjusted models, compared to those in the unfavourable lifestyle group, individuals in the moderate lifestyle group (Hazard Ratio (HR) 0.73 ) and favourable lifestyle group  had lower risk of all-cause mortality. A similar pattern was observed for cardiovascular related mortality and non-cancer/non-cardiovascular related mortality. There was no association of lifestyle with cancer-related mortality. Stratified analysis indicated larger effect sizes among males, those ≤ 73 years old and among those in the aspirin treatment group.In a large cohort of initially healthy older people, reported adherence to a healthy lifestyle is associated with reduced risk of all-cause and cause-specific mortality. The incidence of chronic disease increases with age and are major contributors to mortality amongst older individuals . CardiovCommon unhealthy lifestyle behaviours, such as smoking , high alPrevious studies have suggested that a combination of healthy lifestyle behaviours incurs the strongest benefit in promoting healthy longevity  and reduCurrently, the studies underpinning health guidelines and lifestyle recommendations for the wider population are mainly derived from middle-aged cohorts. The few studies focussing exclusively on older people have limited sample-sizes and/or focussed exclusively on all-cause mortality while neglecting to investigate the impacts on cause specific mortality , 13, 14.The Aspirin in Reducing Events in the Elderly (ASPREE) study, a cohort of initially healthy older people, is uniquely suited to investigate whether a combination of healthy lifestyle behaviours in community-dwelling older people is associated with a prolonged life span and reduced cause specific mortality .This current analysis is based on data from the Australian participants in the ASPREE , and ASPREE trial sub-sets: the ASPREE-eXTension (ASPREE-XT) study -17 and tclinicaltrials.gov on 24/12/2009 (NCT01038583).Following completion of the ASPREE clinical trial (2010 – June 2017), the ASPREE-XT observation follow-up period commenced (ongoing). Data in this analysis includes those collected up to the participants’ second annual ASPREE-XT visit (2020). ASPREE and ASPREE-XT were approved by the local ethics committees and is registered on The ALSOP sub-study is a longitudinal cohort study involving approximately 90% of the Australian participants in ASPREE and ASPREE-XT. Details of the ALSOP study methodology and baseline characteristics have been published elsewhere . ParticiThe lifestyle score was constructed based on four modifiable lifestyle factors  known to be associated with chronic disease onset , 19-23. Full details of the assessment of the lifestyle factors and classification is described in detail in Table S1. Briefly, baseline smoking status was categorised as either current or no current smoking (including former smokers). A previous study using ASPREE data found moderate alcohol consumption to be associated with a reduced risk of CVD and all-cause mortality, as reported previously , 24. In Methodological details on the ascertainment of all-cause and categorisation of cause-specific mortality have been published , 17 and 2 test, analysis of variance (ANOVA) or Kruskal- Wallis test, as appropriate.Participants were included in the current analysis if they had completed both baseline and year-three ALSOP questionnaires . BaselinCox proportional hazards models were used to estimate hazard ratios (HRs) and 95% confidence intervals (CIs) for the association between the lifestyle score and all-cause and cause-specific HRs for each of cancer, CVD and ‘Other’ mortality. Competing risk Kaplan–Meier curves were plotted to illustrate the cumulative effect of lifestyle categories on all-cause and cause-specific mortality. Analyses were adjusted for age (continuous), sex , aspirin treatment allocation (100mg enteric coated aspirin/placebo) (model 1), education (≥ 12-years/< 12-years), living status , and socioeconomic status  (model 2). Full details on the ascertainment of these study measurements have been described in detail previously , 18.2), baseline diagnosis of type-2 diabetes (yes/no) and baseline diagnosis of hypertension (yes/no) and aspirin treatment allocation. Interaction was tested by including a cross-product term along with the main effect terms in the models. In exploratory analyses, we investigated the association between the individual lifestyle factors with all-cause and cause-specific mortality and combinations of individual lifestyle factors with all-cause mortality.To investigate whether a priori selected factors modified the association between the lifestyle score and all-cause mortality, we performed analyses stratified by median age (≥ 74/<74 years), sex , education (≥ 12-years/< 12-years), Body Mass Index (BMI) . A two-sided A total of 11,340 Australian participants were included in the current analysis, and followed for a median of 6.8 years  years. At study entry, the median age was 73.9 (IQR 71.7–77.3) years and 54.2% were female. A total of 702 (6.2%) participants died during the follow-up period.Baseline characteristics of included participants according to the lifestyle score categories as well as by all-cause mortality are shown in A higher proportion of individuals with diagnosed vascular risk factors , who were on antihypertensives and statins, who were pre-frail/frail, with depressive symptoms and/or higher BMI, waist circumference and systolic blood pressure were in the unfavourable lifestyle group. There were no differences across lifestyle groups based on aspirin treatment allocation. Description and prevalence of the lifestyle factors in the population are shown in Table S1. With regard to adherence to healthy lifestyle factors, 97.5% reported no current smoking, 22.3% reported moderate alcohol consumption, 33.3% met the ‘healthy diet’ criteria and 66.9% of participants reported engagement in weekly moderate/vigorous physical activity.p-trend < 0.0001  and physical activity , with non-significant trends for alcohol consumption  and diet . Similar trends were observed for CVD mortality and ‘Other’ mortality.Further analyses according to different combinations of two, three and four healthy lifestyle factors prevalent in at least 2% of the population compared to no or one healthy lifestyle factors are shown in Table S7. None of the observed associations were as protective of all-cause mortality as the combination of 4 factors .Table S8 shows results of the stratified analyses by selected health, demographic and anthropometric factors on the association between the lifestyle score categories and all-cause mortality. No statistically significant differences were observed in the associations between the healthy lifestyle score categories and all-cause mortality by age, sex, education, BMI, diabetes, hypertension and aspirin treatment. Nonetheless, trends indicate larger effect sizes among males, those at or below the median age of 73-years-old and among those in the aspirin treatment group.Results from the various sensitivity analyses are presented in the Tables S9-S12, and page 14 of the supplement. Briefly, excluding former smokers/drinkers (Table S9), the alternative categorisation of alcohol consumption (supplement p 14) and the alternative lifestyle score (Table S10 and S11) did not alter the results. Multivariable adjusted HRs on the association between the year-three lifestyle score categories and risk of all-cause mortality remained equivocal (Table S12).In this cohort of 11,340 community dwelling healthy older Australians, we examined the association between a healthy lifestyle score and all-cause mortality, cancer-related mortality, CVD-related mortality and ‘other’ causes over a median follow-up time of 6.8 years . We found that a healthy lifestyle score at baseline, comprising of four common and potentially modifiable lifestyle factors  was associated with prolonged lifespan in a dose-response relationship, such that each additional lifestyle factor was associated with a 16% lower risk of all-cause mortality, 25% lower risk of CVD mortality and 22% lower risk of ‘Other’ mortality. The current data found no association between lifestyle groups and cancer-related mortality. The benefits of a healthy lifestyle on all-cause mortality had larger effect sizes among males, those ≤ 73 years old and among those in the aspirin treatment group, although interactions were not statistically significant.This is one of the largest and most comprehensive studies, conducted exclusively within community dwelling older people, reporting the association between a lifestyle composite score based on adherence to international health behaviour recommendations and all-cause plus cause-specific mortality. The results are largely in agreement with previous reports investigating different combinations of healthy lifestyle characteristics. A similar study of older Chinese people  reported that, compared to those without any unhealthy factors, those who had a high BMI, poor sleep, unhealthy diet, no physical activity, consumed alcohol and currently smoked were 1.34  times more likely to die from any cause over a ten-year follow-up period . AnotherResults reported here and previously, provide compelling evidence to suggest that individuals reporting a healthy lifestyle in older age have a significantly reduced risk of earlier mortality. The results also demonstrate that current international recommendations for moderate physical activity, no smoking, a healthy dietary pattern and moderate alcohol consumption may still provide a useful predictor of longevity among this older aged cohort.We found no relationship between a healthy lifestyle on risk of overall cancer-related death, which is contrary to findings previously reported in the HALE study as well as among studies of younger cohorts . AlthougSome methodological points may impact the conclusions of this study. In order to construct the healthy lifestyle score, we dichotomised each lifestyle factor according to pre-defined cut-off points. Different threshold values may have resulted in different risk estimates. However, the choice of cut-off was largely based on national and international public health recommendations -28. In sOwing to the absence of baseline dietary data, we utilised year-three dietary data as an alternative replacement within the baseline lifestyle score. It is not certain whether dietary behavior had significantly changed over this three-year period. Dietary changes can occur in older people due to factors such as oral health, income, marital status, medication or change of residence . NonetheFinally, given we do not have detailed information about mid-life lifestyle behaviour in the ASPREE cohort, we cannot confirm whether observed associations are not driven by behaviour earlier in life. Healthy lifestyle behaviours in older age may reflect a long-standing approach to healthy living which, in turn, may be driving these observations. Our results still, however, highlight the benefits of identifying healthy lifestyle factors as predictors of likely future mortality, even among already healthy older people.There are several strengths of our study. ASPREE is a well characterised, large and contemporary cohort of older people who had reached age 70-years or more in relatively good health . FurtherThere are also several potential limitations. First, the ASPREE cohort is comprised of initially healthy volunteers for a clinical trial who are more likely to be attentive to maintaining a healthy lifestyle, hence, may represent a healthier sample of older people compared with the general population. Second, the cohort is largely Caucasian, educated and drawn from communities with access to universal healthcare as reflected by the extensive use of preventive medications including statins (in 30%) and antihypertensive agents (in 51%). Therefore, our results may not be applicable among other socioeconomic and ethnic groups as well as among those residing in lower-to middle-income countries. Third, due to the progressive nature of noncommunicable disease leading to death, with declining function often preceding and possibly influencing lifestyle behaviour, we cannot rule out reverse causality as a partial explanation for these observations. Nonetheless, although survivor bias is a common limitation in healthy cohort studies, our censoring of death events at three-years may also help to mitigate reverse causality.Finally, although potential confounders were considered in multivariable analyses, residual confounding cannot be ruled out. Furthermore, other unmeasured lifestyle and environmental factors may also play a role in determining risk of death. However, demonstrating that these four common lifestyle behaviors are associated with prolongation of an individual’s lifespan remains an important public health message.In a well-characterised population of healthy older people, moderate exercise, a healthy dietary pattern, moderate alcohol consumption and non-smoking was associated with 44% reduced risk of all-cause mortality, when compared to those complying to ≤ 1 healthy lifestyle factor. Previous multi-lifestyle interventions among healthy older people has proven beneficial in reducing risk for CVD and cognitive decline -37, wher


Heavy drinking in women is known to adversely affect pregnancy and fertility. However, pregnancy is a complex process, and the adverse effects of ethanol on pregnancy does not mean that ethanol will have adverse effects on all stages from gamete to fetal formation. Similarly, the adverse effects of ethanol before and after adolescence cannot be generalized. To focus on the effects of prepubertal ethanol on female reproductive ability, we established a mouse model of prepubertal ethanol exposure by changing drinking water to 20% v/v ethanol. Some routine detections were performed on the model mice, and details such as mating, fertility, reproductive organ and fetal weights were recorded day by day after discontinuation of ethanol exposure. Prepubertal ethanol exposure resulted in decreased ovarian weight and significantly reduced oocyte maturation and ovulation after sexual maturation, however, normal morphology oocytes with discharged polar body showed normal chromosomes and spindle morphology. Strikingly, oocytes with normal morphology from ethanol exposed mice showed reduced fertilization rate, but once fertilized they had the ability to develop to blastocysts. RNA-seq analysis showed that the gene expression of the ethanol exposed oocytes with normal morphology had been altered. These results show the adverse effects of prepubertal alcohol exposure on adult female reproductive health. Research on alcoholism is of wide concern because of its negative impact on many facets of life and physical health –5, incluIn prepubertal female ovaries, primordial follicles initiate growth to primary follicles and grow gradually, but they go to atresia before the antral follicle stage due to the lack of gonadotropins. After estrus, some follicles gradually mature and excrete MII-stage oocytes, following the change of gonadotropin secretion in waves. The developmental potential of oocytes before puberty is lower than that of oocytes from sexually mature individuals , 16. To Three-week-old C57BL/6J female mice were purchased from SPF (Beijing) Biotechnology Co., Ltd. The mice were raised in cages of five and divided into two groups. One was the control group (CtrL), the other was the 3-week ethanol exposure group (EtOH) by replacing drinking water with 20% v/v ethanol.The blood glucose of all mice was measured, and the mean-values of blood glucose of CtrL- and EtOH- groups were calculated, respectively. The mice with blood glucose levels near mean-value were selected for testing. For glucose tolerance test (GTT) and insulin resistance test (ITT), mice were fasted for 16 h and 6h, respectively, then 2 g/kg glucose or 1 IU/kg insulin was injected in the abdominal hypodermis. Tail blood was collected for blood glucose detection and blood glucose was measured 0 min, 15 min, 30 min, 45 min, 60 min, 90 min and 120 min after injection by a glucometer (Roche).After a 3-week treatment, mice were evaluated for estrous cycles daily for 18 days at the same time by vaginal lavage. The first 6 days of vaginal lavage were the time of acclimation for the mice, the other 12 days of vaginal lavage were used for smears. The smears were observed under a microscope, and classified as proestrus (P), estrus (E), metestrus (M) and diestrus (D) through the proportion of nucleated epithelial cells, cornified squamous epithelial cells and leukocytes .in vitro in M2 medium. The number of germinal vesicle breakdown (GVBD) oocytes was recorded after 2-4 hours and the number of oocytes that extruded polar bodies (PBE) was recorded after 14 hours. Forty-eight hours after i.p. injection with pregnant mare serum gonadotrophin , hCG  was injected. Fourteen hours later, cumulus-oocyte complexes (COCs) were collected from the Fallopian tubes. For IVF, spermatozoa were obtained from healthy adult male mice and kept in HTF  for 40 min. Sperm and COCs were incubated in HTF. After 10-20 minutes of incubation, MII-stage oocytes with normal morphology were selected microscopically and then co-incubated with spermatozoa for 4-6h in HTF. After sperm-oocyte co-incubation, embryos were cultured in KSOM  until development to blastocysts in vitro. The fertilization rates were determined by counting the number of pronuclei observed 8-9 hours post-fertilization. The number of blastocysts was confirmed at 4.5dpc and 5.5dpc respectively. In addition, female mice with vaginal plug after mating with male were used to observe blastocysts in vivo at 3.5 dpc by uterus flushing.The processes followed published protocols . The ovaAfter three weeks of treatment, the female mice were discontinued ethanol exposure, and mated with healthy adult males for 5 days. The dates of females secreting vaginal plugs were recorded. The pregnancy in the mice with vaginal plugs was recorded to calculate the fertility rate, and fetuses and placentas were collected intact using scissors and forceps for weighing at 19.0 dpc.in vivo were collected 14 hours later from the Fallopian tubes, and digested with 1 mg/ml hyaluronidase . The zona pellucida of MII-stage oocytes matured in vivo were removed with acid Tyrode’s solution . The oocytes without zona pellucida were fixed in 1% paraformaldehyde with 0.15% Triton X-100 and 3 mM dithiothreitol on glass slides. The slides were dried at room temperature and blocked with 1% BSA for 1 h, then incubated in anticentromere antibodies,  at 4° C overnight. After washing, the slides were incubated with secondary antibodies Cy5 (1:200) for 1 h at room temperature. The nuclei were stained with DAPI at room temperature for 15 min. DABICO was added and coverslips were used for lightly covering the samples.The processes were performed as described previously . MII-staMII-stage oocytes were collected and fixed in 4% paraformaldehyde for 20 min. After incubation in 0.1% Triton X-100 for 20 min, the oocytes were transferred to 1 mg/ml BSA for 1 h. The processes above were performed at room temperature. Then the oocytes were incubated with α-tubulin antibody at 4° C overnight, and nuclei were stained with DAPI. Finally, samples were placed on the slides and DABICO was applied. All the specimens were observed under a Zeiss LSM 780 confocal laser-scanning microscope.RNA-seq was performed by Annoroad Gene Tech. (Beijing) Co., Ltd. The MII-stage oocytes were collected by superovulation from 3-week-old mice, 6-week-old control mice and ethanol-exposed mice in tubes with lysing reagent (30 oocytes per tube). All the collected oocytes had obviously extruded polar bodies. Ethanol-treatment did not stop during superovulation in the ethanol-exposed group. Amplification was conducted by the Smart-Seq2 method. After library preparation, PerkinElmer LabChip GX Touch and Step OnePlus Real-Time PCR System were introduced for library quality inspection, and PE150 sequencing was performed using the Illumina HiSeq platform. The experiments of each group were repeated three times. The RNA-seq data were available under NCBI SRA accession number PRJNA719111.Data from repeated experiments were analyzed by independent sample T test by SPSS software. P<0.05 was considered a significant difference. The area under the curve (AUC) of GTT and ITT were calculated with GraphPad Prism version 6.01 for windows, GraphPad Software, La Jolla California USA. Correlation analysis was performed on the gene expression levels of each group by SPSS.To understand the effect of prepubertal chronic ethanol exposure on adult reproduction, we established a prepubertal ethanol exposure female mouse model by changing the 3-week-old mice’s drinking water to 20% v/v ethanol for 3 weeks. After a 3-week ethanol treatment, mice went through prepuberty, and the weight of ethanol-treated mice (n=11) was significantly lower than that of the control mice (n=16) .The GTT and ITT were also performed to make comparison between the control and ethanol-exposed groups. GTT showed that the glucose level at 30-min post-injection was significantly higher in ethanol-treated mice  , and ITTTo further understand the state of prepubertal ethanol exposed mice, the estrus cycle detection was carried out after 3-week of ethanol exposure by vaginal smears. Vaginal smears were examined for 18 consecutive days. The first 6 days of vaginal lavage were the time of acclimation for the mice, the other 12 days of smears were used to record the estrus cycle of model mice (n=17) and control mice (n=16). The results showed that the prepubertal ethanol-exposed female mice had a disordered estrus cycle, and most of the time the ethanol-exposed mice were in metestrus and diestrus . This suAfter a 3-week ethanol exposure, the mice reached puberty. The ethanol exposure was stopped immediately, and the mice were mated with normal adult males. Females were observed daily for the presence of vaginal plugs, and mice with vaginal plugs were recorded as successful mating. The production of offspring was recorded as being fertile. The results showed that the fertility rate of prepubertal ethanol-exposed female mice decreased significantly compared with control mice . After dIn vitro culture of GV-stage oocytes taken from ovaries showed no significant difference in GVBD rates, but the PBE rate was significantly reduced  and the oocytes with normal MII-stage morphology from ethanol exposed mice (EtOH-6-week). Comparing CtrL-6-week-group and EtOH-6-week-group, GO analysis showed that there were 234 GO terms which were significantly enriched P<0.05, . KEGG anThe GO and KEGG analysis showed that the state of oocytes was changed by ethanol treatment, but the intensity of the change was difficult to predict. To understand the damage caused by 3 weeks of ethanol treatment, RNA-seq was performed on oocytes from normal female mice (CtrL-3-week) at 3 weeks of age without ethanol treatment and sexual maturation. Bivariate analysis was used to analyze the correlation between any two of CtrL-3-week, CtrL-6-week, and EtOH-6-week . Some stAlcohol exposure has adverse effects on the reproductive system of females and males , 23, andIn vitro fertilization of MII-stage oocytes of prepubertal ethanol exposed mice showed that the fertilization rate decreased significantly but once fertilized, the zygotes developed normally into the blastocysts in vitro. In addition, we were able to obtain normal blastocysts in vivo from prepubertal ethanol-exposed mice. Oocytes with normal morphology from ethanol exposure mice were affected in terms of binding with sperm, while fertilized eggs could develop into blastocysts. In addition, our RNA-seq analysis showed that the status of the oocytes with normal morphology after ethanol exposure had been altered, although not dramatically. If ethanol had a serious impact on oocytes, it would not be possible to restore fertility in a short period of time, since the oocyte growth takes a relatively long time. Lack of dramatical damage provided a prerequisite for the gradual fertility restore to normal in 4 days from discontinuation of ethanol exposure. There were some differences with previous results. Previous studies had shown that ethanol exposure in vivo lead to very poor oocyte quality, because these studies did not distinguish between morphologically normal and abnormal oocytes. This study focused on oocytes with normal morphology after ethanol treatment, which led to a difference in the results.In addition, prepubertal ethanol exposure affects the growth and development of ovaries and maturation of oocytes. In spite of this, some oocytes with normal morphology could be obtained from ethanol exposed mice. These oocytes had normal chromosomes and spindles. Follicles undergo initial recruitment and cyclic recruitment during their growth. The initial recruitment begins after the mouse is born, and the cyclic recruitment refers to the process by which follicles respond to changes in gonadotropins during the estrus cycle after puberty. The developmental potential of oocytes before puberty was lower than that after sexual maturation. The estrus cycle of prepubertal ethanol exposure mice was disordered, and the disorder mainly appeared as stagnation in metestrus and diestrus period and was basically not in estrus period. This state of estrus disorder indicated that female mice did not have the ability to initiate cyclic recruitment. We suspected that ethanol exposure caused the retardation of reproductive development of prepubertal female mice. That was, after 3 weeks of ethanol-treatment, even if the mice have reached adult age, their physical condition was still not ready to initiate follicle cyclic recruitment. In other words, the state of oocytes from prepubertal ethanol-exposed mice should be between a poor state of prepuberty and a healthy state of adulthood.In fact, this concept was difficult to verify. In addition, the effect of ethanol on prepubertal females was not only developmental delay, but there were still other damages as well. We aimed to using RNA-Seq and bivariate analysis to determine the correlation between oocytes from prepubertal, adult, and ethanol-exposed mice. Notably, we found that the correlation coefficient between CtrL-3-week and CtrL-6-week was lower than that of the two other combinations. That was to say that there might be some correlation between ethanol injury and the loss of developmental potential without experiencing sexual maturation. So, it was concluded that prepubertal chronic ethanol exposure resulted in delayed oocyte development.Our ethanol exposure protocol was to replace the daily drinking water of mice with 20% ethanol. This method involved less human intervention in mice compared with other operational schemes such as injection and gavage which causes emotional stress including fear and resistance. Therefore, the obtained reproductive results in the present study could be more reliable. Cebral et al. defined 5% as low chronic ethanol consumption and 10% as moderate doses of ethanol, while suggesting that 10% could result in many poor eggs , 27. In Our study showed that prepubertal chronic ethanol exposure affected female fertility, but it was possible to select good quality MII-stage oocytes by morphology. Studies had shown that different genetic backgrounds produce different individual differences in alcohol consumption , so our


Regarding stress surrounding the pandemic, the median IES-R showed mild distress in 16 (11%), moderate distress in 7 (5%), and severe distress in 4 (3%). The COVID-19 pandemic lockdowns had a negative impact on patients’ mental health with close to 20% of the patients reporting at least moderately depressive symptoms and 19% reporting at least mildly distressful symptoms.The COVID-19 pandemic had significant impacts on the mental and visual health of patients. This cross-sectional, survey-based, multicentric study evaluates the state of mental and visual health among patients with chronic ocular diseases such as glaucoma, neovascular age-related macular degeneration, diabetic retinopathy, or chronic uveitis during the lockdown period of the COVID-19 pandemic. Mental health was assessed using three questionnaires: the Patient Health Questionnaire-9 (PHQ-9), the Impact of Event Scale-Revised (IES-R), and the National Eye Institute Visual Function Questionnaire-25 (VFQ-25). A total of 145 patients completed the questionnaires. The PHQ-9 showed that most respondents ( The 2019 coronavirus (COVID-19) pandemic emerged as a global crisis, affecting every aspect of human life . NumerouDuring the pandemic, numerous changes were implemented in patient care and health services to adhere to global and local public health guidelines . In ordeIndividuals with chronic diseases represent a vulnerable population due to their health comorbidities and reliance on healthcare services. Chronic ocular diseases encompass a broad spectrum of conditions affecting visual health and have also been correlated to mental health disorders such as depression. For example, diabetic retinopathy is associated with all-cause and cardiovascular mortality, depression, eating disorders, and several forms of cognitive impairment . PatientTherefore, this study aims to explore the specific challenges faced by patients with chronic ocular disease to better understand the interplay between their visual health and mental wellbeing in the context of the COVID-19 pandemic. This study seeks to shed light on the mental health repercussions and psychological distress faced by vulnerable patients at risk of vision loss. Understanding these challenges will help guide patient care and the development of targeted interventions and support systems to mitigate adverse mental health outcomes in the setting of a global health crisis.This cross-sectional study assessed the mental and visual health of patients during the COVID-19 pandemic using validated questionnaires. Patients at greater risk for loss to follow-ups were targeted, using the following criteria for the selection of patients: adult patients (≥18 years old) capable of providing informed consent, at least 3 visits in ophthalmology per year since February 2018, having a scheduled follow-up in March 2020, and a confirmed diagnosis of at least one ocular disease, including chronic glaucoma, wet AMD requiring anti-vascular endothelial growth factor (VEGF) injections, PDR, or chronic uveitis treated with systemic immunosuppressants or corticosteroid drops  at least twice daily. Patients were selected based on the presence of chronic ocular conditions regardless of mental health history. Patients from two university tertiary care centers were included . Data collection included age, sex, systemic health conditions, underlying ocular condition, visual acuity, and intraocular pressure of both eyes at the final follow-up before the initial pandemic lockdown on 13 March 2020. Patients were contacted over the phone to complete the questionnaires between June 2021 and December 2021. To be included in the analysis, patients needed to have completed at least one questionnaire. This study was approved by the ethics board of each center.Patients were contacted via phone, and informed consent was obtained verbally. To obtain informed consent, patients were first contacted, and if they agreed to be part of the study, the questionnaire was given 48 h later in order to give them the time to withdraw from this study. To assess the mental and visual health of patients, a combination of validated questionnaires was used based on the respondent’s preferred language . The Patient Health Questionnaire-9 (PHQ-9)  was usedThe PHQ-9 has been recognized as a reliable and valid tool for screening major depressive disorder . The PHQThe IES-R is a valid tool to assess the response to a traumatic event . Scores The VFQ-25 is a comprehensive general and visual health questionnaire that assesses different aspects via subscales for general health, general vision, ocular pain, near activities, distance activities, social functioning, mental health, role difficulties, driving, color vision, peripheral vision, as well as a composite score . Each ist-test, analysis of variance (ANOVA), Mann–Whitney U test, or Kruskal–Wallis test as appropriate for continuous variables and chi-square analysis for categorical variables. Shapiro–Wilk test and Q-Q plots with 95% confidence intervals were used to test for normality of distribution in continuous variables. Spearman’s rho was used to evaluate correlation between different questionnaire scores. A Spearman’s rho of 0.00 to 0.29 was deemed a negligible correlation, 0.30 to 0.49 a weak correlation, 0.50 to 0.69 a moderately strong correlation, 0.70 to 0.89 a strong correlation, and 0.9 to 1.0 a very strong correlation  PHQ-9 score was 3  (range: 0–46). Most respondents (p > 0.05) between disease categories . Furthermore, as illustrated in p < 0.001). There was also a lower VFQ-25 score in patients with the presence of distress symptoms compared to those without .Regarding stress surrounding the pandemic, the median IES-R was 11 5, 18] (range: 0–46). No significant distress was found in 117 (81%), mild distress in 16 (11%), moderate distress in 7 (5%), and severe distress in 4 (3%). There were 11 (8%) who had scores ≥33, indicating a probable diagnosis of PTSD . A total of 101 patients (70%) answered the optional items, which did not differ by principal disease category , may have affected the IES-R scores of patients as public health policies were changing. One of the major limitations is not having a baseline response rate prior to the pandemic and no available data regarding patients’ previous mental health history. This study is also subject to possible response bias and recalls bias because questionnaires were administered more than a year after the beginning of the pandemic. Finally, this study did not investigate the patients’ perspective regarding their ocular condition or their appreciation of the impact the pandemic has had on their eye care. Additionally, to the best of our knowledge, no studies have looked into the impact of telemedicine on the mental health of patients followed in ophthalmology during the COVID-19 pandemic.This study highlights the negative psychological impacts of the COVID-19 pandemic on patients with chronic ocular conditions, with close to one in five patients reporting experiencing depression and/or some level of distress. This study also found an overall association between vision-related quality of life and mental health parameters regardless of disease category. Consequently, mental health assessment and screening using questionnaires such as PHQ-9 should be considered for patients with chronic ocular disease, especially in the setting of a pandemic or any other public health crisis. Additional research is needed to develop and implement resources for mental health adapted for patients with ocular comorbidities.


KRAS-variant, an inherited germline mutation, benefit from cetuximab, we evaluated KRAS-variant patients in RTOG 0617.RTOG 0617 was a phase III randomized trial for patients with unresectable stage IIIA/IIIB non–small cell lung cancer comparing standard-dose (60 Gy) versus high-dose (74 Gy) radiotherapy and chemotherapy, plus or minus cetuximab. Although the study was negative, based on prior evidence that patients with the P value <0.05 was considered significant.From RTOG 0617, 328 of 496 (66%) of patients were included in this analysis. For time-to-event outcomes, stratified log-rank tests and multivariable Cox regression models were used. For binary outcomes, Cochran—Mantel–Haenzel tests and multivariable logistic regression models were used. All statistical tests were two sided, and a KRAS-variant, and overall survival rates were similar between KRAS-variant and non-variant patients. However, there was a time-dependent effect of cetuximab seen only in KRAS-variant patients—while the hazard of death was higher in cetuximab-treated patients within year 1 , death was lower from year 1 to 4 . In contrast, in non-variant patients, the addition of cetuximab significantly increased local failure .A total of 17.1% (56/328) of patients had the KRAS-variant patients, there is potential impact of cetuximab for this genetic subset of patients. In contrast, cetuximab seems to harm non-variant patients. These findings further support the importance of genetic patient selection in trials studying the addition of systemic agents to radiotherapy.Although an overall survival advantage was not achieved in KRAS-variant is the first functional, inherited miRNA-disrupting variant identified in cancer. Our findings support that cetuximab has a potentially beneficial impact on KRAS-variant patients treated with radiation. The work confirms prior evidence that KRAS-variant patients are a subgroup who are especially sensitive to radiation. These findings further support the potential of this class of variants to enable true treatment personalization, considering the equally important endpoints of response and toxicity.The  Unfortunately, results from this trial were negative, with the highest median survival of 28.7 months achieved in the standard-dose arm without cetuximab. Both the radiation dose escalation and cetuximab results crossed futility boundaries and there was evidence that the dose escalation was harmful to patients, likely due to increased heart dose  is a cancer type with many such efforts, as local failure (LF) remains unacceptably high, especially in patients with advanced stage disease . RTOG 06art dose , and thaart dose .The negative results from RTOG 0617 were not dissimilar from several other phase III trials combining cetuximab with radiotherapy, including one in esophageal cancer , the short nature of which was hypothesized to potentially be due to the short course of cetuximab in the study (8 weeks). The positive response was found to be strongest in the HPV+ patients, and hypothesized to be due to the potential immune stimulating effects of cetuximab  and skin reaction inside the portal (18.4% vs. 15.6%). This was in contrast to non-variant patients, who had lower toxicity with radiation and cisplatin which was significantly increased with the addition of cetuximab; including mucositis  and skin reaction inside the portal . Both KRAS-variant and non-variant patients had significantly increased skin reactions outside of the portal with the addition of cetuximab, which was not correlated with cetuximab response in that trial.Because of the benefit of cetuximab for t manner . The timetuximab , as thisppressed . In addiKRAS-variant patients respond to cetuximab in combination with radiation and cisplatin with improved outcomes, but with increased radiosensitivity at baseline  or homozygous (GG) for the variant allele.All patients included in this study were consented for the clinical trial RTOG 0617, which was conducted in accordance with recognized ethical guidelines. All patient samples were received from the RTOG/NRG sample repository as anonymized blood or buffy coat samples. All DNA was isolated using Qiagen standard protocols, evaluated for purity, and quantified and prepared for This analysis was performed using data from all eligible patients from RTOG 0617. Eligible patients who did not receive radiation or received ≤51 Gy were excluded from this analysis. All analyses were based on “as-treated” populations for this secondary analysis. Specifically, patients were considered as receiving a “standard” dose of radiation if they received more than 51 Gy but less than or equal to 66 Gy and receiving a “high” dose of radiation if they received more than 66 Gy; patients were considered as receiving cetuximab if they received cetuximab during concurrent therapy or beyond, and receiving no cetuximab if they did not receive any cetuximab or received only the loading dose.The efficacy outcomes included in this analysis included OS, local failure (LF), distant failure (DF), and PFS, as defined in KRAS-variant was evaluated by (i) comparing KRAS-variant and non-variant patients after stratifying by radiation dose and cetuximab; (ii) multivariable regression model, including KRAS-variant, radiation dose, as well as their interaction, after stratifying by cetuximab; (iii) multivariable regression model, including KRAS-variant, cetuximab, as well as their interaction, after stratifying by radiation dose. In addition, the predictive value of the KRAS-variant was evaluated through subgroup analysis within KRAS-variant and non-variant patients, by comparing radiation dose groups (stratified by cetuximab) or cetuximab groups (stratified by radiation doses). These analysis methods were used for all clinical outcomes studied. For time-to-event outcomes, stratified log-rank tests and multivariable Cox regression models were used. For binary outcomes, Cochran–Mantel–Haenzel tests and multivariable logistic regression models were used.The prognostic value of the P values were reported accordingly. Multivariable Cox regression model was used when evaluating the prognostic value of KRAS-variant.OS, PFS, time to LF, and time to DF were analyzed as time-to-event data and calculated from the date of randomization to the date of failure or, if no failure, the date of respective competing event or last follow-up. The rates of OS and PFS were estimated using the Kaplan–Meier method and compared using the stratified log-rank test . The ratBest observed response was analyzed as a binary outcome, whether complete or partial response was achieved versus otherwise. Safety was analyzed as a binary outcome, whether worst treatment-related toxicity was grade 3 or higher versus otherwise. Cochran–Mantel–Haenzel tests and multivariable logistic regression model were used to analyze these two endpoints.P value <0.05 was considered statistically significant. SAS v9.4 (SAS Institute) was used for all statistical analyses. Representativeness of the study participants are shown in All statistical tests were two sided, and a KRAS-variant genotyping in this study are available on request from the corresponding author.The data on KRAS-variant genotyping and/or completed treatment, we first evaluated whether patients who were included in the analysis were systematically different from those who were not.KRAS analysis appeared to receive 3D-conformal radiotherapy (CRT) versus intensity-modulated radiotherapy (IMRT) more frequently than those excluded (P = 0.0006). In addition, as all patients included in the analysis received at least 51 Gy, this was significantly different from the excluded patients and cohort overall, where 14.9% and 5% did not receive this amount of radiation, respectively (P < 0.0001). Otherwise, patients included or excluded were similar.As only a subset of patients had samples available for P = 0.81)), PFS , LF  or distant metastasis (DM) . However, patients included tended to have slightly better observed responses with more complete/partial responses  there were no significant differences, except there were more PET-staged non-variant patients than KRAS-variant patients . There were also similar stage groupings between KRAS-variant and non-variant patients. These findings are consistent with prior findings for patients with KRAS-variant NSCLC  of included patients had the nt NSCLC .KRAS-variant and non-variant patients, considering as assigned and as treated . These findings suggest that the high-dose treatment negatively affected patients regardless of KRAS-variant status, but that the negative impact may have been especially impactful for KRAS-variant patients.For non-variant and KRAS-variant status and time. In RTOG 0617, cetuximab was delivered for 15 or 16 weeks. In non-variant patients, there was no difference in OS or time-dependent OS between the cetuximab versus no cetuximab-treated group, but the cetuximab-treated group had a numerically nonsignificant shorter median survival . However, unlike non-variant patients, in KRAS-variant patients treated with cetuximab the worse outcome seemed to occur primarily in year 1, with fewer KRAS-variant patients treated with versus without cetuximab alive at the end of year 1 . We found that for KRAS-variant patients there was a time-dependent effect of cetuximab on OS; while the hazard of death was significantly greater in year 1 for KRAS-variant cetuximab-treated patients , the risk of death was significantly lower for KRAS-variant cetuximab-treated patients from years 1 to 4 ] ] . In contpatients .KRAS-variant with grade 3+ treatment-related toxicity. Overall, patients with the KRAS-variant had similar toxicity rates as non-variant patients, with no differences comparing hematologic toxicity, pulmonary toxicity, or esophagitis after adjusting for (stratifying by) radiation dose and cetuximab [P = 0.69]. Comparing the high-dose versus the standard radiation dose treatment arms, dose was not found to be a significant predictor of increased toxicity using logistic regression models (KRAS-variant patients in the high-dose arm versus the standard-dose arm (40.0% vs. 22.2%).We next evaluated the association of the n models . HoweverKRAS-variant patients (P = 0.0003), but was not specific to hematologic, pulmonary, or esophagitis. Both non-variant and KRAS-variant patients had significantly increased toxicity when cetuximab was delivered with treatment . This difference appeared to be driven primarily by DF, with a significantly higher rate of DF in KRAS-variant versus non-variant patients with squamous NSCLC . Although due to the small sample size, it was not possible to meaningfully evaluate the impact of cetuximab on this subgroup, cetuximab did appear to improve OS for KRAS-variant patients comparing survival at year 1 for cetuximab-treated versus non–cetuximab-treated patients (92.3% vs. 37.5%), but the overall impact was not significant (HR = 0.94, CI = 0.35, 2.54, log-rank test P = 0.95; Because cetuximab has been generally used as a radiosensitizer for squamous cell cancers, and the prior benefit of cetuximab for patients with KRAS-variant patients with NSCLC benefitted from cetuximab. Although there were a relatively small number of KRAS-variant patients in each arm due to the multiple interventions in this trial, and we did not see an OS advantage for KRAS-variant cetuximab-treated patients, there were some interesting findings in this analysis. First, there was what appeared to be a time-dependent benefit of cetuximab for KRAS-variant patients with NSCLC, who exhibited significantly improved OS from year 1 to 4 when they received cetuximab. However, KRAS-variant patients appeared to have toxicity and do poorly with treatment intensification, including radiation dose escalation, or cetuximab therapy, with poor survival in the first year of treatment across groups. In addition, and in agreement with findings in other trials combining radiation and cetuximab, non-variant patients had significantly worse outcomes with the addition of cetuximab, with worse local control, indicating that cetuximab may be especially harmful to non-variant patients.In this phase III trial of radiation dose escalation in locally advanced NSCLC with or without cetuximab, we performed a hypothesis-driven subgroup analysis to determine whether inherited KRAS-variant patients had an OS benefit that lasted for 2 years. In RTOG 0617, cetuximab was given for approximately twice as long, or 16 weeks, and the OS benefit, after year 1, lasted for 4 years. One could hypothesize that the longer cetuximab treatment in 0617 extended the survival benefit for KRAS-variant patients. It is also interesting that in 0617 patients with squamous cell NSCLC with the KRAS-variant appeared to have significantly worse outcomes than non-variant squamous patients, with a potential benefit of cetuximab. This might better represent the population treated in 0522 and may be the group with the greatest benefit from the combination of cetuximab and radiation. Unfortunately, this subgroup in 0617 was too small for proper analysis of cetuximab benefit in this study.The time-dependent effects of cetuximab seen in this trial are similar to the findings in RTOG 0522, where patients with HNSCC were treated with 8 weeks of cetuximab and the subgroup of KRAS-variant patients appear to be at risk of significant toxicity from both dose-escalated radiation as well as cetuximab. In 0617, these sensitivities may have led to their early death, explaining the poor survival in KRAS-variant patients in year 1 in both the dose-escalation group as well as the cetuximab-treated group. Although excessive death was not seen in KRAS-variant patients treated in 0522, the tissues treated in 0522 differ from 0617, with the heart as the suspected tissue at risk for the high toxicity and death and poor survival seen in 0617. The ability to truly differentiate death due to treatment versus death due to cancer remains a true challenge in clinical trials, especially those in difficult to control cancers where aggressive treatment is necessary to try to improve survival. NSCLC is one such cancer, and has the additional risk associated with treatment near to organs that are sensitive to radiation yet critical for survival.Another important finding from RTOG 0522 further validated in this study is that KRAS-variant patients, nor do we fully understand the cause of increased radiosensitivity with the delivery of cetuximab, even after using this treatment in combination with radiation in numerous clinical trials. One simple explanation for radiosensitization in both KRAS-variant patients and with cetuximab may be that disruption of normal signaling through the EGFR-KRAS axis, as is found in patients with the KRAS-variant (KRAS-variant patients could also be due to elevated TGF-B1 as reported previously (let-7 levels found in these patients (let-7 is known to also known to be a critical player in the radiation response (We do not yet fully understand the mechanisms leading to radiosensitivity in -variant  and with-variant . Howevereviously  or perhapatients , as let-response . The impKRAS-variant is the first example, are a compelling class of biomarkers that may ultimately help us dramatically improve our approach to cancer therapy. Through their ability to identify patients with altered response to cancer treatment, as well as altered toxicity to cancer treatment, they could enable a future where patient treatment selection is genetically based, and that in addition to cancer control, treatment decisions will include the equally important endpoint of patient toxicity.Although the results from this study are unlikely to lead to changes to improve outcome for patients with NSCLC, there are important takeaways from this analysis, and the findings offer some hope for the future. The miRNA-based germline pathogenic variants of which the inherited Supplementary Data Table 1Endpoints evaluated in RTOG 0617Click here for additional data file.Supplementary Data Table 2Representativeness of Study ParticipantsClick here for additional data file.Supplementary Data Table 3KRAS Analysis Inclusion StatusClick here for additional data file.Supplementary Data Table 4Pretreatment Characteristics by KRAS Analysis Inclusion StatusClick here for additional data file.Supplementary Data Table 5Best Observed Response by KRAS Analysis Inclusion StatusClick here for additional data file.Supplementary Data Table 6Worst Overall Treatment-Related Adverse Event by KRAS Analysis Inclusion StatusClick here for additional data file.Supplementary Data Table 7Pretreatment Characteristics by KRAS-variant or non-variant statusClick here for additional data file.Supplementary Data Table 8Overall SurvivalClick here for additional data file.Supplementary Data Table 9Overall Survival for non-variant and KRAS-Variant Patients by RT Level AssignmentClick here for additional data file.Supplementary Data Table 10Overall Survival within non-variant Patients Cetuximab AssignmentClick here for additional data file.Supplementary Data Table 11Local Failure within KRAS Variant Patients by As-Treated CetuximabClick here for additional data file.Supplementary Data Table 12Worst Treatment-Related Toxicity By KRAS GenotypeClick here for additional data file.Supplementary Data Table 13Worst Treatment-Related Toxicity Logistic Regression Model of KRAS and As-Treated RT InteractionClick here for additional data file.Supplementary Data Table 14Worst Treatment-Related Toxicity within Non-variant Patients By Cetuximab AssignmentClick here for additional data file.Supplementary Data Table 15Worst Treatment-Related Toxicity within KRAS-Variant Patients By CetuximabClick here for additional data file.Supplementary Data Table 16Overall Survival Squamous Cell Variant Patients OnlyClick here for additional data file.