Accumulating evidence suggests that externalising problems are consistently associated with alcohol use behaviours, but findings are inconsistent regarding the role of internalising problems. We investigate whether externalising and internalising problems are associated with problematic drinking in mid-adulthood, and whether potential associations are modified by age, sex and cohort.n = 17,633) and 1970 British Cohort Study  recruited new-borns in Great Britain in a single week in 1958 and 1970. Mental health was assessed with the Rutter Behaviour Questionnaire at ages 7, 11, and 16 in NCDS58 and ages 5, 10 and 16 in BCS70. Problematic drinking was measured with the CAGE questionnaire at age 33 in NCDS58 and age 34 in BCS70, and the AUDIT scale at age 44/45 in NCDS58 and age 46 in BCS70. Latent scores of externalising and internalising problems were added chronologically into lagged logistic regression models.The National Child Development Study , and internalising was a protective factor . Associations between early life mental health and mid-adulthood problematic drinking did not differ by developmental timing but were stronger in males.Our study provides new insights on links of externalising and internalising difficulties with alcohol use and has implications for public policy in the UK.The online version contains supplementary material available at 10.1007/s00127-021-02063-3. Alcohol contributes substantially to the global burden of disease, not only through alcohol use disorders (AUD) but also via other disease consequences resulting from alcohol use, such as injuries and suicide, cardiovascular disease and cancer . KnowledTheoretical and empirical research supports the idea that alcohol problems in adulthood have their roots in early life . One lonA series of factors  may contribute to the inconsistencies, but no definitive conclusions can be drawn as those factors were rarely examined within the same study . ExternaThe present study extends the current literature by investigating the association of early life externalising and internalising problems with problematic drinking in mid-adulthood, and whether these associations are modified by age, sex and cohort using two successive nationally representative longitudinal British birth cohorts.We used data from two British birth cohorts born 12 years apart in England, Scotland, and Wales: the 1958 National Child Development Study (NCDS58) and the 1970 British Birth Cohort (BCS70). NCDS58 recruited 17,633 babies born in one week in March 1958 with 10 waves of data collected through 2013. BCS70 recruited 17,568 babies born in one week in April 1970 with 9 waves of data through 2016. Details about both cohorts are available elsewhere .Tools used to assess externalising and internalising problems, alcohol outcomes and potential confounding factors varied across waves and cohorts. To explore the potential cohort effect in the associations between externalising and internalising problems and problematic drinking, variables that were consistently collected across cohorts were retained and harmonised for analysis.Externalising and internalising problems were measured with the parent rated Rutter Behaviour Questionnaire . In NCDSCut down/Annoyed/Guilty/Eye-opener) questionnaire is a four-item self-report screening instrument for detecting alcohol problems [The CAGE  at age 33, and 16,336  at age 44/45. In BCS70, it is 16,655  at age 34, and 16,593  at age 46. Due to attrition over more than 40 years (ESM Table 6), which is known to erode sample representativeness if left unchecked, multiple imputation (MI) was implemented. One hundred fifty imputed datasets were generated using chained equations using a two-stage calculation –49, and P value. In the second stage, interactions of externalising and internalising problems at each age with sex and cohort were investigated. This was achieved by adding interaction terms into the previous models. The above models are displayed in notation form in ESM Text 2.The analysis proceeded in two stages. In the first stage, lagged logistic regression was carried out by adding externalising scores, internalising scores and corresponding confounders chronologically to explore the direction of the association and the potential critical period of externalising and internalising problems. For example, externalising and internalising scores at age 5/7 and confounders measured at birth and age 5/7 were first included in Model 1, and then externalising and internalising scores and time-varying confounders at age 10/11 were further added in Model 2. Last, externalising and internalising scores and confounders at age 16 were added in Model 3. To investigate potential critical periods of experiencing externalising and internalising problems with respect to their associations with later problematic drinking, post hoc comparisons were conducted. For instance, comparisons were made to see whether the coefficient of externalising scores at age 5/7 in Model 1, at age 10/11 in Model 2 and at age 16 in Model 3 were equivalent to each other. Potential increases in Type I error, due to multiple comparisons, were corrected using Bonferroni corrections at a threshold of 0.017 (0.05/3) for the A series of sensitivity analyses were carried out to assess the robustness of our results from various perspectives. The rationale of each analysis is articulated in ESM Text 3, and results are presented and discussed where appropriate.As shown in Table E value ranged from 1.20 to 1.29, indicating that an unmeasured confounding factor would still need to be associated with externalising or internalising problems and problematic drinking at a risk ratio of 1.20–1.29 to nullify the observed association to 1. The strength needed to render the confidence interval to include 1 was smaller (1.08–1.22).Externalising and internalising problems at each age in childhood and adolescence were associated in opposite directions with problematic drinking, and these associations persisted across mid-adulthood  with problematic drinking did not differ between externalising and internalising problems, and that the strength of the associations between externalising or internalising problems measured at age 5/7, age 10/11 and age 16 and problematic drinking in adulthood did not differ by age.p = 0.028), with the association between externalising problems and problematic drinking at age 33/34 being observed only in males. An interaction between internalising problems and sex was detected at both age 33/34 (p < 0.001) and age 45 (p = 0.026), with the association being stronger in males.Interactions of externalising and internalising problems with sex predicting the probability of problematic drinking were mainly detected at age 16. These are plotted in Fig. We did not observe interactions between externalising or internalising problems and cohort regarding their associations with problematic drinking at any age (ESM Table 8).By analysing data from two population-based prospective British birth cohorts born 12 years apart, we found that externalising problems across childhood and adolescence acted as a risk factor for problematic drinking in adulthood. In contrast, a negative association between internalising problems and problematic drinking was observed, which indicates that those experiencing internalising symptoms in childhood or adolescence were less likely to exhibit problematic drinking in mid-adulthood. We did not detect a critical period of experiencing externalising and internalising problems between early childhood and mid-adolescence regarding their associations with problematic drinking. Strength of the associations did not differ significantly across cohorts but were stronger in males.Existing evidence is quite inconsistent regarding the association between early life internalising problems and alcohol use behaviours in adulthood , 52. ThiThe association between early life internalising problems and later alcohol use behaviours may vary across the life course, as determinants and patterns of drinking typically change with development. Adolescence is a period of dramatic changes physically, psychologically and socially; drinking in this period is strongly socially-driven, greatly affected by social norms, peer influences and parents’ monitoring , 56. ForFurthermore, the association between early life internalising problems and alcohol use behaviours in adulthood may vary across populations. The proposed internalising pathway to alcohol use/disorder suggests that people with internalising problems use alcohol to self-medicate or gain acceptance by peers . HoweverOur results support the possibility that not adjusting for externalising problems could introduce residual confounding in the association between internalising problems and alcohol outcomes. The observed opposite direction of the associations of externalising and internalising problems with alcohol outcomes has also been found in previous studies where externalising and internalising problems were adjusted for simultaneously , 65–68. Results from post hoc comparisons did not identify any critical periods in childhood or adolescence when experiencing mental health problems showed a stronger or weaker association with problematic drinking in adulthood. Few studies have had sufficient longitudinal data to explore the developmental timing of externalising or internalising behaviours and their potential impact on alcohol use behaviours in mid-adulthood. Individuals with externalising problems may actively self-select into environments and peer settings where alcohol use is common, consistent with their dispositions, and they may be reinforced reciprocally by these environments . Person-Another finding worth our attention is how the associations between externalising and internalising problems and problematic drinking differed significantly across sex but not cohorts. Born 12 years apart, participants from NCDS58 and BCS70 were raised during a time of shifting social environments towards alcohol , 79. ThoIf causal, our findings have implications for public policy. First, externalising problems instead of internalising problems might be a better target for early life intervention to alleviate alcohol burden in adulthood in the UK context. Though the high co-occurrence between externalising and internalising problems is well-established , 93, mosStrengths of our study include the availability of two well-characterised prospective national birth cohorts with repeated measures of mental health that span childhood and adolescence and measures of problematic drinking in early adulthood and midlife. This adds to the scarce evidence on how far into adulthood the pathway from early mental health to alcohol use persists. Furthermore, considering the high rate of co-occurrence between externalising and internalising problems in childhood and adolescence , 42, 97,r > 0.95 for NCDS58, r > 0.92 for BCS70), externalising and internalising problems in our study should be interpreted with caution, especially when comparing the results with other studies. Third, the short version of AUDIT used in our study increases the rate of false positives in detecting problematic drinking , was calculated [E value in our study  indicates that the association of the unmeasured confounder with problematic drinking, conditional on all the covariates included in the model, would need to be stronger than the observed associations of externalising problems with problematic drinking (1.06–1.11) in our study. Thus, it could be argued that the observed associations were less likely to be fully nullified by unmeasured confounding factors, as the externalising pathway is, up to now, shown to be the most robust pathway [Several limitations should also be considered when interpreting our findings. As in any other longitudinal survey, selective attrition is unavoidable. In this instance, about 60% of the original sample was retained by age 46 in both cohorts. By making use of the abundant information collected in both longitudinal studies , we impulculated . However pathway . On the  pathway , 103, pa pathway . HoweverBy utilising two British birth cohorts, we found that externalising problems in childhood and adolescence were positively associated with problematic drinking across mid-adulthood while internalising problems were negatively associated with problematic drinking, and the results were robust in a series of sensitivity analyses. We also found no evidence for a particular critical period for experiencing externalising and internalising problems in childhood or adolescence that was uniquely important as predictors of problematic drinking in mid-adulthood. The stability of these associations across two UK cohorts born 12 years apart indicates the developmental nature of the association between externalising and internalising problems and problematic drinking. More evidence is needed to examine whether the detected sex differences in the associations persist for more recent generations. Our study provides new insights on links of externalising and internalising problems with alcohol use in the UK. If causal, our findings could inform design of early life interventions to reduce alcohol burden in adulthood.Supplementary file1 (DOCX 114 KB)Below is the link to the electronic supplementary material.


Cognitive biases can adversely affect human judgment and decision making and should therefore preferably be mitigated, so that we can achieve our goals as effectively as possible. Hence, numerous bias mitigation interventions have been developed and evaluated. However, to be effective in practical situations beyond laboratory conditions, the bias mitigation effects of these interventions should be retained over time and should transfer across contexts. This systematic review provides an overview of the literature on retention and transfer of bias mitigation interventions. A systematic search yielded 52 studies that were eligible for screening. At the end of the selection process, only 12 peer-reviewed studies remained that adequately studied retention over a period of at least 14 days  or transfer to different tasks and contexts (one study). Eleven of the relevant studies investigated the effects of bias mitigation training using game- or video-based interventions. These 11 studies showed considerable overlap regarding the biases studied, kinds of interventions, and decision-making domains. Most of them indicated that gaming interventions were effective after the retention interval and that games were more effective than video interventions. The study that investigated transfer of bias mitigation training (next to retention) found indications of transfer across contexts. To be effective in practical circumstances, achieved effects of cognitive training should lead to enduring changes in the decision maker's behavior and should generalize toward other task domains or training contexts. Given the small number of overlapping studies, our main conclusion is that there is currently insufficient evidence that bias mitigation interventions will substantially help people to make better decisions in real life conditions. This is in line with recent theoretical insights about the “hard-wired” neural and evolutionary origin of cognitive biases. People constantly form judgments and make decisions, both consciously and unconsciously, without certainty about their consequences. The decision to take another job, to start a relationship, or to visit a friend is generally made without knowing beforehand how internal and contextual “success-factors” will develop, or what will happen when these decisions are really carried out. When making these kinds of decisions our thinking is characterized by systematic distortions that often seem to violate the rules of logic and probability. These violations may be manifested in cognitive biases  knowledge on the existence and nature of cognitive biases has appeared insufficient to mitigate them  and retention or transfer . These elements were adapted to the respective databases. For example, the search string used in Scopus was: (title-ABS-key [“bias mitigation” OR debiasing] AND ALL [retention OR transfer]). The field specifications were made to search for papers that deal with retention and transfer, within the more general topic of debiasing.near transfer,” for instance to another set of similar test items, situation assessments, or obvious questions that are considered to measure cognitive bias, were excluded. So, asking subjects to assess situations, how they would behave or if they think that they have learned, were not considered as sufficient measures of transfer. Finally, only primary experimental studies were used.Studies where included if they investigated the effectiveness of cognitive bias mitigation interventions. Cognitive biases had to be investigated explicitly, while studies investigating an improvement of performance in a broader sense were excluded  compared to that of a related video in mitigating the bias blind spot, confirmation bias and fundamental attribution error (or correspondence bias). While both the game and the video reduced all biases, the game reduced the biases more, both right after the game and eight weeks later. The second experiment evaluated a different game than Experiment 1 and compared it to a related video. The targeted biases were anchoring, representativeness, and social projection. Like in the study of Clegg et al.  that was used in the first of the two experiments reported by Morewedge et al. , in comparison to a related video that taught the same cognitive biases and an unrelated video that did not teach any biases. All five games and the related video mitigated the biases right after the intervention, while three games outperformed the related video. After eight weeks the overall mitigation effects remained, while now four video games outperformed the related video. The second experiment evaluated the three games that outperformed the video right after the intervention. Two of these outperformed the related video right after the intervention; however, after 12 weeks no game produced better debiasing results than the related video anymore. The related video outperformed the games in teaching the definition of biases, underlining once again that knowledge of biases is not sufficient to achieve substantial bias mitigation in people. The unrelated video was added to investigate whether there was an effect of mere practice, irrespective of its kind. This effect was indeed observed: after eight weeks participants were less biased than right after watching the unrelated video. Therefore, the mere effect of practice seemed to have a “debiasing” effect. However, this effect was smaller than the debiasing effect of the games. To achieve bias mitigation, games appear more effective than videos.The article on retention by Rhodes et al.  discusseThe article on retention by Lee et al.  investigyes,” while this was only the case for 32% of the participants who received awareness training for these biases . This may imply that the training affected transfer to some extent. That is, the statistical biases were mitigated the most from the  debiasing training. Yet, for the other biases, improvement after either the awareness or the analogical training was not found.The article on retention by Aczel et al.  evaluateA summary of all 12 studies reviewed in this paper is presented in Bias mitigation training has only practical value when the bias mitigation effects of the intervention are retained over time and transfer across contexts. To explore the evidence for the effectivity of bias mitigation interventions, we performed a systematic literature review on the retention and transfer of bias mitigation interventions. More specifically, this review investigated the  evidence that bias mitigating interventions yield stable effects that transfer to situations beyond the mere training context. Our main conclusion is that the scientific evidence for both retention and transfer of bias mitigating interventions is scarce. While our literature search initially yielded 52 articles after duplicates were removed, only 12 articles (including 15 experiments on retention and three on retention and transfer) remained after careful selection on relevance and methodological considerations. In addition, all studies  involved real behavior transcending the specific training conditions, problem structures, and domains  are typically explained with the Dual Processing Model  issue. All stimuli entering the nervous system affect its structure and thereby its connectionist properties. So, unlike computer software, once information has entered the brain, it cannot simply be erased or made undone. Relating this example of neural information processing to biases: irrelevant information or counter-productive information (which has been provided) is always associatively integrated into the brain's physical-chemical structure. It is captured in the brain's neural circuitry and thus may (associatively) affect a following judgement or decision. This means that judgement and decision making is by definition always affected to some extent by persisting (“anchoring”) effects of information that has been processed before. Biased decision making may then occur when irrelevant or misleading information associatively interferes with the reasoning process. Examples of these neurally inherent biases are for instance Anchoring bias  characteristics of  neural networks, biases may also originate from ingrained evolutionary  heuristics that promoted the survival of our ancestors who lived as hunter-gatherers for hundreds of thousands of years in close-knit groups , striving for admiration by peers , or herd tendencies like following or copying the opinions or behavior of the majority  or biases following from our limited “higher” cognitive functions, like statistics, probability reasoning or calculation, which have been developed only very recently in evolution  situations and contexts. This counts especially for the biases that originate from the previously mentioned inherent or structural characteristics of biological neural networks. The reason for this is that these neural biases are inextricably linked to the structural system properties of biological neural networks, like the human brain. Because of these inherent properties it is impossible for the brain to correctly adhere to principles like: “environment or context in which people make decisions instead of trying to directly improve their thinking capacities. For example, one could stimulate or impose the use of certain very strict working methods or aids with which the ingrained tendency to biased thinking can be prevented or circumvented. Examples of these are checklists or premortems (e.g., countering optimism bias by imagining what could make a project go wrong; Klein, However, the notion that many biases arise from more or less hard-wired brain mechanisms, means that mitigating them will always be an “uphill-battle” requiring substantial motivation, effort and perseverance. Therefore, we suppose that the most promising way of dealing with biases may be to improve the The original contributions presented in the study are included in the article/JK conceived the original idea and supervised this study. JG searched the literature and wrote the initial draft paper. AT critically reviewed the draft manuscript. All authors were actively involved in the revisions of the original drafts and in writing the final version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Background: Patients suffering from complete colonic aganglionosis (TCA) require the best surgical care possible. Only a few studies reported J-Pouch repair as the primary reconstructive surgery in TCA patients. This study adds to the current literature a thorough clinical and functional outcomes group. Methods: Between 2011 and 2021, medical records of Hirschsprung disease (HD) patients who underwent J-Pouch reconstruction during infancy (n = 12) were reviewed. In close follow-up, bowel function and satisfaction with operation results were evaluated. The median age at the time of J-Pouch reconstruction was 16 months, and covering ileostomies were closed four months later. There were no postoperative problems. After the final repair, Pouch-related problems (PRP) occurred in 27% of the children and were treated conservatively. There was no histological evidence of pouchitis in any of the individuals. The median 24-h stooling frequency was 4–5 at the latest follow-up 51 months following enterostomy closure. Conclusions: The current study’s findings support the existing literature and advocate for J-pouch repair in TCA patients. However, more research will be needed to determine the best time to undergo pouch surgery and ileostomy closure in TCA patients. Complete colectomy is the only curative treatment for total colonic aganglionosis (TCA). However, the goal of such intensive surgical treatment must be followed by a reconstruction that leads in the long term to complete continence. Ravitch originally described colectomy with continent repair in adult patients in 1951 utilising a straight ileoanal anastomosis . HoweverPublications in paediatric and adolescent surgery suggest that in patients with TCA, a straight ileoanal anastomosis is preferable to forming a pouch . The keyThe study followed the principles of the Helsinki Declaration and was approved by the local institutional review board Hamburg ethics committee (2021-300128-WF). The study’s goal was to characterise the outcomes of complete colectomy followed by primary J-pouch formation during childhood. From January 2011 to December 2021, all TCA children were screened in hospital records from the paediatric surgery departments at the University Center Hamburg-Eppendorf and Altona Children’s Hospital Hamburg.The medical patient records were analysed to look at the presentation, diagnostic studies, operative approach , complications, functional results, growth, and electrolytes status. Regular outpatient clinic visits were used to collect follow-up data. There were also questions about weaning age, urine continence, stool frequency, abdominal distension, nutritional status, and satisfaction with functional and cosmetic results. All data from eligible patients was recorded into a standardised spreadsheet. The study included all TCA patients who had undergone reconstructive surgery with a total colectomy and ileoanal J-pouch reconstruction. Patients who had J-Pouch reconstruction for reasons other than TCA and all patients with rectosigmoid HD were excluded. The primary goal was to evaluate faecal incontinence and pouchitis and enteritis-like symptoms. Presenting symptoms, perioperative complications and postoperative outcome were evaluated as binary variables . Patient demographics were calculated as median with maximum and minimum values given as total range. Statistical analysis was performed descriptively using Microsoft Office Excel .Protective enterostomies were performed on all patients involved in this study (mean age six months). Total colectomy was performed via laparoscopy or laparotomy, followed by a transendorectal pull-through technique, depending on the number of previous procedures and associated intra-abdominal adhesions.The colon was surgically prepared with caution to protect the ileocolic artery, which is critical in supplying the ileoanal J-pouch. The pouch reconstruction was done extracorporeally. The pouch length was determined using the criteria for visceral surgery; however, the required lengths were lowered to ensure healthy growth of the residual small intestine. As a result, we evaluated a pouch length of 5 cm to a maximum of 7 cm in infancy. Because of its smaller branches than a typical GIA-stapler, the J-pouch was created with the help of the EndoGIA.The final rectal resection and ileoanal J-pouch anastomosis were performed transanally, with the dentate line and transition zone preserved using an anal retractor. TCA does not require a complete mucosectomy, which is needed for familial adenomatous polyposis (FAP) or ulcerative colitis (UC), lowering the risk of postoperative sensory problems. A Foley catheter was inserted, and was kept for several days after surgery since nerve irritation of the hypogastric plexus could cause temporary bladder palsy.For four weeks, all patients were followed up weekly. Particular emphasis was paid to assessing the progression of anastomotic stricture, which may necessitate the use of bougienage at an early stage if necessary.From 2011 to 2021, nine boys and three girls received subtotal or total colectomy for long-segment HD followed by J-pouch repair . Given tNine children (81%) showed typical symptoms during their neonatal period. Four children presented with constipation and five with failure to thrive throughout their first year of life, and they included not passing meconium, a distended abdomen, and bilious vomiting . TCA wasReconstruction was performed on patients ranging in age from 3 months to 5 years, with a mean of 16 months, and all patients underwent a temporary double-lumen ileostomy. Stoma closure was performed at an average age of 4 months  after primary repair in the current cohort. There were periodic follow-up assessments following the primary repair, particularly for stool frequency and continence, urinary continence, nutrition, failure to thrive, and vitamin, electrolyte, and iron status. However, two of the patients are currently in school, while the rest are still in infancy.All of the patients in our sample were over the third percentile in terms of body weight and size . PatientAfter closure of the protective stoma, we observed a stool frequency of 15–20 bowel movements per day, resulting in electrolyte and fluid loss as well as skin irritation in the perianal region. To manage bowel movements, some patients required loperamide medication. Overall, all patients had decreased stool rates of roughly 4–5 bowel movements per day throughout time. Today, six of nine patients above the age of 3 years are entirely continent, and two of 9 are completely continent during the day. At the age of 8 years, the patient with Trisomy 21 begins weaning off the diaper. In addition, two children are too young to be weaned from diapers .Seven individuals had their vitamin and electrolyte levels checked regularly. While vitamin D replacement was required for five children, vitamin A, B12, and E replacement were required for four, three, and one child, respectively.Urinary electrolytes were tested on a regular basis and revealed that sodium excretion was reduced in all instances. As a result, sodium was administered orally to newborns. Electrolytes were supplied utilising glucose electrolytes supplements by an oral rehydration solution after infancy. Again, after stoma closure, the need for sodium substitution decreased. Only one patient out of seven has normal sodium and potassium excretion values. Four patients continue to receive oral rehydration solution. In four children, iron substitution was required.While perioperative mortality was 0%, perioperative morbidity was similarly low, with an average hospital stay of 11 days following reconstruction surgery. All perioperative issues were stoma-related, with four patients experiencing stoma prolapse, one experiencing parastomal hernia, and one experiencing mild stoma retraction.Two significant issues emerged following stoma closure. They resulted in the most common reason for hospital readmission after surgery: (1) Sphincter achalasia caused defecation problems in six children, as evidenced by recurring indications of bowel obstruction or enteritis; (2) fluid loss and electrolyte imbalances, particularly during bouts of enteritis. Both issues, however, were strongly related to age, with the highest hospitalisation rate observed in the second year of life. Nine of the eleven children were affected. On the other hand, conservative treatment with intravenous fluids and antibiotics was sufficient to treat all children.Another common complaint was increased stool frequency, which caused sleep issues and significant perianal skin maceration in seven children immediately after stoma closure.In addition, three occurrences of pouch-related issues were noted (PRP). In one child, PRP relapsed within a year, while the other child developed transient PRP. Both patients, however, were successfully treated with antibiotic treatment, as an immunological explanation for PRP was considered unlikely. There was no histological evidence of pouchitis in any of the individuals. Complications did not necessitate revisions. There were no reports of anastomotic leakage, stricture, or fistula. One postoperative ileus was handled conservatively. Three months after reconstruction, a small submucosal defect was discovered during rectoscopy in one individual .This is the largest single-center dataset of TCA patients describing the outcomes of J-Pouch repair. We operated on 12 TCA patients and closely monitored their progress. With the exception of two children, one with trisomy 21 and one girl just four weeks after total colectomy and J-Pouch reconstruction, it reveals that all children were completely weaned from diapers before their third birthday and had a reasonable continence rate overall. The average follow-up period was 51 months, ruling out any conclusions about long-term outcomes. However, a multicenter study of patients repaired using different procedures found similar results, with continence rates of 86 percent in the pouch group compared to 48 percent in the ileoanal anastomosis group 12 months after surgery . The risWhen ileoanal anastomosis was compared to J-pouch reconstruction, regardless of diagnosis, it was discovered that the J-pouch construction had a higher rate of pouchitis in the first few years after the reconstruction, but continence scores were significantly improved 24 months later when compared to straight ileoanal anastomosis . HoweverIncreased frequency of bowel movements is a serious and possibly debilitating condition after J-Pouch repair . Both toAside from the immediate postoperative outcomes, the medium and long-term out-comes of pouch repair appear to favor J-Pouch reconstruction, which is consistent with the development in adult visceral medicine . HoweverThis is unfortunate, because pouch reconstruction provides the highest quality of life, with an average of 5–6 bowel movements per day and complete continence in more than 90% of adults . After total colectomy for TCA, the J-Pouch should be the recommended surgical technique. The primary reason for this is that it is simple to build and, in the long run, produces functional results equivalent to other procedures .A problem that has yet to be overcome is determining whether a colectomy and J-pouch reconstruction are appropriate for the patient and when an ileostomy closure is the best option. We believe that all patients receiving TCA should have a prophylactic ileostomy to reduce postoperative complications such as anastomotic leakage. However, our findings show that early ileostomy closure is associated with more hospitalizations, resulting in insufficient emptying and an increased risk of bacterial over-growth of the small intestine and PRP . This efDue to impaired function, a colectomy with a direct ileoanal repair is not an option in adult visceral surgery. Our findings and the current literature support J-pouch reconstruction in TCA patients. However, more research will be needed to determine the best time to undergo pouch surgery and ileostomy closure in TCA patients.The current study may be limited in accuracy due to missing data in the charts due to the study methodology. Furthermore, the small sample size and the study’s single center retrospective nature limits the conclusions derived from the current study.


The prepared Mg2+@PAM/GG hydrogel was characterized by morphology, water vapor loss, swelling ratio, rheological properties, tensile mechanical, biocompatibility, and flow cytometry study. The results show that Mg2+@PAM/GG hydrogel’s mechanical strength could be enhanced by the dual network structure and physical crosslinking agent Mg2+ ions. In addition, the tension strength of Mg2+@PAM/GG hydrogel is obviously increased from 86 to 392 kPa, the elongation at break increased from 84 to 231%, and crosslinking density N increased from 4.3 to 7.2 mol/m3 compared with pure GG hydrogel. The cumulative release curve of Mg2+ ions shows that the multiple release mechanism of Mg2+ ions belong to non-Fick’s diffusion. Meanwhile, in vitro experiments show that Mg2+@PAM/GG double network hydrogel has favorable proliferation and an NF-κB pathway inhibition property for fibroblast cells. Finally, the healing effect of the Mg2+@PAM/GG was evaluated in a rat full-thickness burn model. The animal study demonstrates that Mg2+@PAM/GG could accelerate the healing efficiency in case of the sustained-released Mg2+ ions in wound beds. Considering this excellent performance, this convenient prepared hydrogel has great potential as a commercial application for skin full-thickness burn healing materials.To accelerate serious skin burn wound healing in a convenient manner, an interpenetrating network of hydrogel consisting of gellan gum and polyacrylamide was synthesized by chemical crosslinking and Mg Burn wounds are serious devastating traumas with significant cost for both individuals and the health care system. Among all types of burns, third-degree burns, also known as full-thickness burns, destroy the full thickness of the skin, provoking immediate cell death and matrix destruction with the most devastating damage at the surface of the wound. Skin grafting and dressing is the traditional treatment for full-thickness burns but often with delayed wound healing, tissue necrosis, and scar formation. To solve the challenge associated with severe burns, various active hydrogel biomaterials have been developed to provide a beneficial environment for wound healing .An ideal hydrogel for severe burn repair should satisfy those requirements, include keeping the wound moist, absorbing excess exudate, covering the sensitive underlying tissue without adherence, inhibiting inflammation development, and actively accelerating the wound healing process at a low cost. Gellan gum (GG) is a kind of naturally anionic polysaccharide that is produced by the bacterium sphingomonas elodea . It is e2+ ion immersion techniques. This precursor hydrogel was prepared by heated acrylamide and GG monomer solutions, in which acrylamide was crosslinked by a chemical agent, and GG was crosslinked by metal Mg2+ ions, respectively. The poly-acrylamide (PAM) provides the high swelling ratio with good mechanical properties to make up the insufficiency of GG , MgCl2 (M8266), acrylamide (A8887), N,N′-methylenediacrylamide , and ammonium persulfate  were purchased from Sigma-Aldrich. All reagents were purchased from Sigma-Aldrich without further purification.GG  for 12 h to obtain the Mg2+@GG/PAM IPN hydrogel. Finally, the Mg2+@GG/PAM hydrogel was freeze-dried for future use.The interpenetrating network hydrogel of GG and PAM was synthesized by mixing the following homogeneous ingredients: The synthesis of Mg2+@GG/PAM hydrogel, pure GG hydrogels were also prepared by adding equal weight GG  in deionized water.To validate the subsequent performance of Mg−1. The wave number resolution was selected as 2 cm−1.FT-IR: The FT-IR spectra of hydrogels (lyophilized status) were measured using a Bruker instrument. Spectra ranged from 4,000 to 500 cm2+@GG/PAM hydrogel (lyophilized status) was observed by SEM (Philips LEO1530 VPSEM). The chemical element distribution of Mg2+@GG/PAM hydrogel was analyzed by EDS.Scanning electron microscopy (SEM) and energy dispersive spectrometer (EDS) analysis: The cross-section morphology of Mg2 atm.Thermo gravimetric analysis (TGA): TGA (Mettler-Toledo) of hydrogels (lyophilized status) was executed with the temperature ranging from room temperature to 700°C with a heating rate of 10°C/min under NTension tests: The hydrogels (hydrogel status) tensile property was executed by a universal testing instrument  with a strain rate of 50 mm/min.Rheological test: The rheological tests of hydrogels (hydrogel status) were executed through a rotational rheomete  operated in 20 mm parallel plate geometry and 1 mm gap distance. First, the gelation behavior of the hydrogel was tested via frequency sweep with a constant strain of 1% by varying amplitude frequency 0.1–100 rad/s. Finally, the strain sweep was tested with the constant frequency of 6.3 rad/s and oscillatory strain from 0.1 to 100%.2+@GG/PAM hydrogel was conducted by the water vapor transmission rate (WVTR), and the detailed operation referred to ASTM standard E96–00 , and the solution was placed under 120 rpm shaking at 37°C for 64 days. At predetermined time points, 4 ml of solution was taken out to measure the Mg2+ release amount, and 4 ml volume of fresh PBS was added. The amount of released Mg2+ was determined using inductively coupled plasma-mass spectrometry (ICP-MS). The cumulative release of Mg2+ was calculated with the following equation: Mg2+ (%) =  × 100%.Mg2+@GG/PAM hydrogel was tested using 3T3 mouse fibroblast cells and RAW264.7 cells. For 3T3 mouse fibroblast cells, the DMEM medium supplemented with 10% calf bovine serum was used as the culture medium. The fibroblast cells (1×105 cells per well) were cultured onto the Mg2+@GG/PAM hydrogel (lyophilized status) surface for 1, 2, and 3 days. At day 3, acridine orange/ethidium bromide  was stained and observed by fluorescence microscope (Olympus TH4-200). Meanwhile, the proliferation ability of 3T3 fibroblast cells was quantified by the MTT -2,5-diphenyltetrazolium bromide) method. The control, GG, GG/PAM, and Mg2+@GG/PAM groups were added with 50 μL MTT solution and incubated 4 h. Then, the absorbance was detected using a microplate reader. The apoptosis of fibroblast cells was detected with flow cytometry by Annexin V-FITC/PI apoptosis kit . For RAW264.7 cells (1 × 105 cells per well), they were cultured in DMEM high-sugar medium onto a plastic plate or Mg2+@GG/PAM hydrogel surface for 72 h to study their anti-inflammatory ability. In addition, lipopolysaccharide (LPS) and PBS were added into the medium to make the concentration 10 mg/ml, respectively. In total, the cells were divided into four groups: control (PBS), LPS, Mg2+@GG/PAM (cultured onto Mg2+@GG/PAM hydrogel), and LPS + Mg2+@GG/PAM (cultured onto Mg2+@GG/PAM hydrogel with LPS added) groups. Then, they were collected, and we conducted the Western blot experiment.The viability and proliferation performance of MgFor protein analysis, fibroblast cells were rinsed in PBS and lysed using radioimmunoprecipitation assay (RIPA) buffer containing 1% (v/v) phenylmethylsulfonyl fluoride . Afterward, the protein supernatant was harvested and detected by the bicinchoninic acid (BCA) protein assay kit . The protein was denatured in a water bath  after adding a protein-loading buffer. The cells lysates were then processed with 12% SDS-PAGE gel electrophoresis at 120 V for 1 h. The polyvinylidene fluoride (PVDF) membrane  was used to load proteins. Then Western blocking buffer was used to treat the PVDF membranes, which were incubated by anti-NF-κB p65 (ab16502) and β-actin (ab115777) primary antibodies overnight at 4°C. The next day, the tris-buffered saline (TBS) containing 0.1% Tween 20 (TBST) buffer was used to rinse the PVDF membranes twice, and then the PVDF membranes were incubated for 2 h by the secondary antibodies  in a Western secondary antibody dilution buffer. The Tannon 5,200 Multi image analysis system  was used to test all blot intensities. The Quantity One software  was used to test band density, and blots were carried out in triplicate.2+@GG/PAM hydrogel. The hydrogels were sterilized by Co-60 irradiation at 10 kGy  before treatment, and antiseptic gauze was placed as a cover over the hydrogels and sewn to the wound to prevent infection. Then, the wound was fastened with an elastic bandage to prevent wound contraction. The animal tissue specimens were obtained after treatment for 7, 14, 21, and 28 days.All animal experimental precepts conformed to the oversight of Guangzhou sport university laboratory animal ethics, Guangzhou, China . The 32 Sprague–Dawley rats  were equally divided into four groups and given pentobarbital sodium through intraperitoneal injection, and the dorsal hair was removed. Deep burn wounds were generated on the dorsal skin using a hot nummular copper block with diameter of 18 mm, which was heated in a 100°C water bath. The copper block was placed on the shaven posterior-dorsum of each rat for 50 s to create a full-thickness burn model. Then, the polypropylene isolation chamber was implanted onto the wounds to inhibit skin shrinkage. The carnosus membrane of wound tissue was removed 24 h later. Then, the deep-thickness wounds were covered with GG, GG/PAM, MgThe tissue surrounding the wounds was obtained 7, 14, 21, and 28 days after implantation of hydrogels. The tissues were fixed with paraformaldehyde for 24 h at 4°C, and hematoxylin-eosin (H&E) staining, Masson’s trichrome staining, and TNF-α immunohistochemical staining were carried out to evaluate wound healing and inflammatory reactions. The histological sections were examined using a stereomicroscope . All samples were performed with at least three wounds per group. For immunohistochemical staining, the tissue paraffin sections were soaked in xylene solution for 15 min; soaked in 70, 80, 90, 95, and 100% alcohol solution for dehydration for 10 min each; and then PBS was used to wash off the extra alcohol. Then, 3% bovine serum albumin was used for nonspecific blocking for 40 min. Anti-TNF-ɑ primary antibody  was incubated overnight and rinsed with PBS buffer three times and then stained with secondary antibody at 37°C for 30 min, then rinsed with 0.01 M PBS buffer three times, about 5 min each time. Finally, a 3,3′-diaminobenzidine hydrochloride (DAB) color reagent kit was used to visualize color.p value was regarded as significant with *p < 0.05, **p < 0.01.All tests were repeated in triplicate unless otherwise stated. Data are represented as average ±standard deviation (SD). Statistical analysis was conducted using analysis of variance (ANOVA) test with GraphPad Prism v8 software. 2+ ions, and the acrylamide molecular chain was chemically crosslinked by N,N′-methylenediacrylamide to form a Mg2+@GG/PAM INP hydrogel. The cross-section morphologies of Mg2+@GG/PAM hydrogel were observed by SEM  is also crucial for wound dressing. As the wound exudate is often generated from the wound surface, timely absorption of the exudate is important to maintain a moist environment and accelerate the wound healing rate . The good mechanical property of the Mg2+@GG/PAM IPN hydrogel is because of the dual network architecture creating the nonlinear tension against deformation, and the structure is similar to biological soft tissues.Good mechanical property is the guarantee for the dressing’s commercial application. The mechanical properties, including rheological property and stress–strain behavior of pure GG and Mgquency ω  and straquency ω  dependedensity N  is calcu2+ ion cumulative release curve at the predetermined time under PBS and pH = 9.0 conditions simulates the slightly alkaline environment in the burn wound. It can be seen from 2+@GG/PAM hydrogel group showed a controlled cumulative release rate of Mg2+ ions in the PBS and alkaline simulative condition. The release mechanism was brought by the ion exchange and the swelling property of the hydrogel. The release experiments show that Mg2+ shows better release performance without initial burst release. This is because Mg2+ ions act as the crosslinking agent of the GG molecular chain versus burst release and swelling. The Mg2+@GG/PAM hydrogel showed the best release performance with a cumulative release rate of 36.8 ± 1.95% in PBS and 32.1 ± 1.86% in pH = 9.0 at 31 days. The slightly declined cumulative release rate in pH = 9.0 might be the cause of the enhanced negative-charged carboxyl group, which future strengthened the interaction between the carboxyl group and Mg2+ ions. To better analyze the release behavior of Mg2+, the zero order release equation, first order equation, Higuchi equation, and Korsmeyer–Peppas model were fitted. The curve-fitting degree was calculated by the regression coefficient (R2), which is listed in R2 value of the four models are higher than 0.9, and n was more than 0.45 in both PBS and alkaline solutions. The results suggest the multiple release mechanism of Mg2+ ions in the simulated environment. In addition, the value of R2 for Korsmeyer–Peppas model is 0.99684, 0.99327, and n is 0.940, 0.979 in PBS and alkaline solutions, respectively. This result indicates that Mg2+ release performance features the drug diffusion and frame erosion release mechanisms and belongs to non-Fick’s diffusion in vitro. This release mechanism might be because Mg2+ ions participate in the skeleton of Mg2+@GG/PAM hydrogel. The good release behavior provides a guarantee for Mg2+@GG/PAM hydrogel to maintain biological activity ions for curing skin burn wounds.The Mg2+@GG/PAM hydrogel in skin cell growth and proliferation, fibroblast cells were incubated on the Mg2+@GG/PAM hydrogel surface for 72 h and then observed by fluorescence microscope. 2+@GG/PAM hydrogels were homo-dispersed and visualized a green fluorescence. This shows that the Mg2+@GG/PAM hydrogels possessed favorable biocompatibility for fibroblast cells, which is essential for cell growth and proliferation . Based on AO-EB fluorescence microscope images, we also calculated the cell proliferation amounts using ImageJ software  and NO in immune cells , Masson’s trichrome, and TNF-α immunohistochemical staining were carried out. For the HE staining . These gels displayed double network behavior for improved mechanical properties compared with their respective single network hydrogels. The INP hydrogels show that the tension strength of Mg


N = 789) were offered an educational video prior to completing an online questionnaire that examined genetic knowledge, the perceived severity of MPS III, perceived risk, and attitudes toward ECS. Outcomes were compared to reference data collected previously in which respondents had been offered an educational text (N = 781). We first again studied the attitudes toward ECS in a smaller educational text group (N = 266) in order to assess whether attitudes had changed over time due to increased media coverage on ECS, which did not reveal any significant changes. Respondents who were offered the video had a better genetic knowledge, perceived MPS III as more severe, perceived their risks higher and were more likely to participate in ECS compared to those who were offered text. Online video may well be used as supportive tool to the genetic counseling process, creating more knowledge on ECS and severe genetic disorders included in preconception screening panels.Preconception expanded carrier screening (ECS) aims to identify couples with an increased risk of having a child with an autosomal recessive (AR) disorder before pregnancy, thereby enabling reproductive choices. Genetic knowledge and experiential knowledge both influence the uptake of ECS. As people in the general public often lack such knowledge, it is essential to provide appropriate and understandable information when offering ECS. This study investigated the effect of an educational video, compared to an educational text, on the knowledge and attitudes toward preconception ECS in the general population. Both the text and video consisted of a brief educational summary on AR inheritance and on the type of disorders included in ECS, with the progressive neurodegenerative condition mucopolysaccharidosis type III (MPS III) as an example. Participants in the reproductive age were invited in collaboration with a research agency. Respondents ( Genetic knowledge and experiential knowledge on disorders included in screening panels both influence the uptake of preconception expanded carrier screening.The use of video for educating the general public on preconception expanded carrier screening and on the disorders included in such tests leads to increased genetic knowledge and may well be used as supportive tool to the genetic counseling process.1., Mucopolysaccharidosis type III (MPS III), Zellweger syndrome, and Tay‐Sachs disease), as advised by the European Society of Human Genetics  disorders are generally born into families with no prior family history for that specific disorder. Carrier couples are thus mostly not aware of their carrier status and increased risk of having affected offspring  and experiential knowledge on the potential impact of a severe genetic disease are important factors determining a person's intention whether or not to participate in ECS programs  were recruited online in October 2018 by TNS Kantar, a Dutch research agency, that provide access to a panel of more than 200,000 individuals who are willing to participate in research in exchange of a small reimbursement. The sample was stratified from their database based on the key demographics gender, socio‐economic status, educational level, and regional area to guarantee a representative sample of the Dutch population. Informed consent was obtained from all respondents. The Medical Ethics Committee of the Amsterdam UMC, the Netherlands, stated that Medical Research Involving Human Subject Act (WMO) does not apply to this study as it concerns an anonymous questionnaire study, and therefore, formal ethical approval was not necessary.The recruiting procedure was identical to the procedure used in a previous study on the attitudes toward preconception ECS in the general Dutch population  were offered an educational video (video group) presenting information on ECS and MPS III based on the educational text offered in the study of Nijmeijer et al. . The dat2.2The educational text, video, and questionnaire were all in Dutch. Supplementary materials for this publication were translated into English.2.2.1A full description of the original educational text as used in the study by Nijmeijer et al.  can be f2.2.2https://youtu.be/V9FKDNF_‐tI, including English subtitles).The video was designed and produced in collaboration with an organization specialized in educational videos in the field of medicine . The script of the educational text was used as frame from which the video was designed. We used informative animations with a voice over to visualize the information on AR inheritance and the ECS test. The general concept of ECS, reproductive options, and the nature and course of MPS III as an example of the type of disorders included in ECS were explained by a medical doctor, supported by visual bullets and animations. MPS III was illustrated by video‐recording of two MPS III patients in their home environment . In addition, extra factual information about the general risk of being a carrier couple (1 in 150) and the general risk of having an affected child with one of the AR disorders included in the ECS test (1 in 600) had been added. The video lasted 6:27 minutes  pregnancy, marital status, and religion were collected. Additionally, three multiple choice questions assessed familiarity with carrier screening, with hereditary diseases and previous experience with genetic carrier testing.2.3.2one partner is carrier for one of the diseases, couples have an increased risk to have a child with that disease’) and was answered on a three point scale . The total score ranges from 0 to 7, at which a higher score indicates a better genetic knowledge  (Appendix The perceived severity of MPS III was assessed by two statements (‘I believe MPS III is a severe disease’ and ‘I believe that the life expectancy of MPS III is very bad (severe patients often die before reaching adulthood)’) and answered on a five‐point Likert scale (1 = Appendix .2.3.4very low to 5 = very high)  were assessed and answered on a five‐point Likert scale .Intention to participate in ECS, which was measured multiple choice .Perceived benefits of (A) and barriers against ECS (B), and freedom‐of‐choice statements (C), measured by using a five‐point Likert scale .Perceived personal consequences of ECS: Considerations regarding test results of ECS (A), Perceived consequences as a carrier (B), and Perceived reproductive choices as a carrier couple (C), measured by using a five‐point Likert scale  and computed to a total sum score (range 0–7). All items containing five‐point scales were transformed to binary coded items  disagree/not disagree/not agree or (very) low/not low and not high, 1 = agree/totally agree or (very)high). The word pairs (domain II) were compromised into three answer categories: 1 =  disagree, 2 = do not disagree/do not agree, and 3 =  agree. First, it was assessed whether sociodemographic characteristics, genetic knowledge, perceived severity of MPS III, perceived risk, and attitudes toward preconception ECS remained the same over time by comparing outcomes of respondents who received the text in 2016 and 2018 by using Mann–Whitney U tests and chi‐square tests. Second, answers on the genetic knowledge test, the perceived severity of MPS III, the perceived risk, and attitudes toward ECS were compared between the video group and the text group by using Mann–Whitney U tests and chi‐square tests to assess the impact of the educational video compared to text. Statistical Package for Social Sciences (SPSS) version 25 was carried out for all statistical analyses . p‐values < .05 were considered statistically significant.Descriptive statistics were performed to describe the sociodemographic characteristics of all groups. Sociodemographic characteristics of the groups were compared by using independent sample t tests for continues data and chi‐square tests for categorical data. The items of the genetic knowledge test were transformed to binary coded items .The sociodemographic characteristics of the text group and video group are presented in Table 3.2p = .223). Moreover, no significant differences between both groups were found in 60 of the 62 items relating all other domains. Participants in the current text group only agreed more often on the statement ‘the carrier test can avoid suffering for future parents’  and that MPS III has a very bad life expectancy .There was no significant difference in the number of respondents expressing an intention to participate in ECS between the groups who received the educational text in the current study and 2 years previously in the study by Nijmeijer et al.  (35.1% a3.33.3.1p < .001).Respondents in the video group scored higher on the knowledge test  compared to respondents in the text group  and more often agreed that MPS III has a very bad life expectancy .The majority of all respondents experienced MPS III as a severe disease. However, respondents in the video group more often agreed that MPS III is a severe disease (83.7%) compared to the text group  and a chance of 1:600 of having a child with a severe hereditary disorder as (very) high .Respondents in the video group and the text group equally perceived a chance of 1:6 of being a carrier of a severe hereditary disease as (very) high (70.3%). Compared to the text group, the video group more often perceived a chance of 1:150 that both partners are carrier of the same disease as (very) high .IILevel of agreement on feelings toward ECS in general and personally considering ECS.The minority of respondents stated that they would participate themselves in ECS. However, respondents in the video group reported more often that they would probably or certainly participate in preconception ECS compared to respondents in the text group  and text group (47.2%) was that they want to spare their child a life with a severe hereditary disorder Table . Out of p < .001).There was a significant difference between respondents in the video group and text group regarding their agreement on most items of the domain ‘perceived personal consequences’ Table . For exa4The purpose of the current study was to assess the impact of an educational video compared to an educational text on the knowledge and attitudes toward preconception ECS for severe childhood onset disorders in the general population. MPS III was used as an example of the type of disorders that are included in ECS panels.First, we show that genetic knowledge and the attitudes toward ECS, as assessed by our questionnaire, had not changed over 2 years' time. Therefore, we can assume that the difference in attitudes between the text group and video group is due to the mode of presenting the information and not to accumulating knowledge and awareness over time.Watching the educational video led to an increased intended uptake of ECS testing. Moreover, respondents who watched the video had an overall more positive attitude toward preconception ECS compared to those who read the text. In addition, participants who were offered the video as source of information scored significantly higher on the genetic knowledge test. This is in line with earlier studies demonstrating the positive effect of using video to increase knowledge of (future) parents on Down Syndrome screening . However, intended participation is not always a good reflection of the actual test uptake, since the actual test uptake may be influenced by other factors such as time and costs  screening programs  and the risk of having an affected child when being a carrier couple (1 in 600) was added. However, respondents in the text group were also informed about these risks as this was included in the questionnaire domain ‘perceived risk’. This may have minimized the influence of this difference in content. In addition, the video comprised additional information as the concept of AR inheritance was explained by animations and the example of the disorders included in the ECS panel, MPS III, was illustrated by video‐recordings of two patients. Therefore, the differences between the text group and video group might be caused by the different methods of presenting information, but also by the additional content.4.2As ECS is currently offered to individuals or couples without an a priori risk for, and limited awareness of the disorders included in the screening panels, it is important to educate the general public about the various aspect of ECS and the type of included disorders. This study shows that an educational online video may well be an effective supportive tool to this end and may be used by health care professionals, including genetic counselors and general practitioners.4.3Although our educational video led to improved genetic knowledge compared to text, we show that surprising misconceptions about ECS may still remain. Further research may examine the effectiveness of different types and combinations of educational tools. This might include a combination of video and text, interactive web‐based learning, whether or not in combination with a brief contact with a trained counselor which might be done in person or by phone or video consultation. We believe that such studies are paramount to determine the optimal way to educate the general public on preconception ECS which is essential for responsible and effective introduction of this type of screening.5The results of our study are in line with the current era of online, visual and concise information retrieval as it shows that the use of video for educating the general public on preconception ECS and on the disorders generally included in such tests is superior to text and leads to increased genetic knowledge. Moreover, this study shows that potential uptake rates of ECS may increase with the use of video as educational tool. The success of preconception ECS programs should not be assessed merely on the uptake of screening but rather in terms of a measure of informed choice of (prospective) parents  does not apply to this study as it concerns an anonymous questionnaire study and therefore formal ethical approval was not necessary. Informed consent was obtained from participants prior to completing the online questionnaire.No non‐human animal studies were carried out by the authors for this article.The data that support the findings of this study are available from the corresponding author upon reasonable request.Appendix S1Click here for additional data file.Appendix S2Click here for additional data file.Appendix S3Click here for additional data file.


OsNAR2.1 line was used as a testing target in rice plants with high nitrogen-use efficiency to study the changes of rice methylation and growth in respond of the endogenous and external nitrogen stress.Nitrogen (N) is an important nutrient for plant growth, development, and agricultural production. Nitrogen stress could induce epigenetic changes in plants. In our research, overexpression of the OsNAR2.1 expression  compared with high parent seed nitrogen (HPSN) in high nitrogen external supply. However, such decreases were not observed in wild type. Furthermore, methylation sequencing results showed that LPSN caused massive gene methylation changes, which enriched in over 20 GO pathways in the filial overexpression line, and the expression of OsNAR2.1 in LPSN filial overexpression plants was significantly reduced compared to HPSN filial plants in high external N, which was not shown in wild type.Our results showed that external N deficiency could decrease seed N content and plant growth of the overexpression line. During the filial growth, we found that the low parent seed nitrogen (LPSN) in the overexpression line could lead to a decrease in the filial seed nitrogen content, total plant nitrogen content, yield, and OsNAR2.1, resulting in a heritable phenotype of N deficiency over two generations of the overexpression line.We suggest that the parent seed nitrogen content decreased induced DNA methylation changes at the epigenetic level and significantly decreased the expression of The online version contains supplementary material available at 10.1186/s12870-021-02953-3. Oryza sativa L.) is a major staple food for a large part of the global population. Nitrogen (N) is an essential macronutrient for plant growth and crop productivity [Rice  improves nitrate and ammonium absorption and increases nitrogen-use efficiency (NUE) in rice cultivation systems [pOsNAR2.1:OsNAR2.1 (Ox1) in Chen et al. (2017) as a testing high N-use efficiency rice and to explore both seed and external N content influence on the growth of rice.OsNAR2.1 is a high-affinity nitrate transporter partner protein which, along with OsNRT2s, plays a central role in nitrate absorption and translocation in rice –30. Prev systems . We usedOur research aims to elucidate the influence of seed N resource on the wild type and high N-use efficiency Ox1 line growth in the both seedling and mature, and the methylation change in response to the different seed N and different external N resource.OsNAR2.1 (Ox1) and wild type of Wuyungeng 7(WYG7) in the field with 300 kg N/ha (+N) and 0 kg N/ha (−N)  and water (−N) for 1 month  in both treatments but showed no difference between each other .In conclusion, the low parent seed nitrogen (LPSN) could lead to the retard of the growth and decrease of biomass, N content of seedling and decrease of We grew WYG7-HN, WYG7-LN, Ox1-HN, Ox1-LN lines in the field with four different N content: 300 (HN), 150 (MN), 75 (LN), and 0 kg N/ha (NN) Fig. a-d. FirsOsNAR2.1 of four plants in the mature stage and found out that the expression of OsNAR2.1 was significantly lower in the WYG7 lines compared to Ox1 lines. However, there was no difference between WYG7-HN and WYG7-LN in all four fields  between WYG7-LN and WYG7-HN, Ox1-LN and Ox1-HN, as WYG7-DMR and Ox1-DMR, respectively, in HN and NN treatments. We found that in HN fields, the WYG7-DMR was 28,880, and approximately 3400 more than 25,546 Ox1-DMR. However, WYG7-DMR was 26,288, 6500 more than 19,765 Ox1-DMR in the NN condition. Meanwhile, there are 46% hyper WYG7-DMRs in the HN field but, 65% hyper WYG7-DMRs in the NN field. On the other hand, 75% Ox1-DMRs are hyper in the HN condition but only 34% are hyper in the NN condition . Both WYG7 and Ox1 lines had over 10,000 DMGs in the HN condition. In the NN treatment, there were over 10,000 DMGs in WYG7, but only around 8000 DMGs in Ox1  visualized their methylation states Fig.  and S8. Y46 Fig. b, OsMADSOsNAR2.1 and OsNRT family genes in Fig. We also visualized the methylation of the OsDRM3 and OsCMT3 was significantly lower in Ox1-LN compared with Ox1-HN and there was no difference in OsMET1, OsDRM2, and OsCMT2  and OsNAR2.1 RNAi knock-down line (RNAi) of Nippobare background with wild type (NP) [OsNAR2.1 only half in Ov199-LN compared with Ov199-HN  , 26 with-HN Fig. . But theine Fig. –12. The OsNAR2.1 was significantly lower in LPSN Ox1 seedlings in N fertilizer condition, but not in N deficiency condition  reported that P deficiency could induce 20% hyper-DMRs in the shoot and 86% hyper-DMRs in the root, and gene ontology enrichment analysis presenting differential methylation in response to low phosphorus was strongly enriched in signaling components . In our To further investigate the methylation influence on the gene patterns, we cluster-analyzed the unique DMGs influence in different N fields and found the number of unique methylation genes in WYG7 and Ox1 in HN condition was similar 4831 and 4617)  is regulated by low phosphorus stress [OsDRM2 and OsCMT2 was significantly reduced in the LPSN Ox1 line. In the N deficiency field, the expression of OsDRM3 and OsCMT3 was significantly reduced in the LPSN WYG7 line. DRM2 is responsible for de novo methylation at all sites [Since methylation changes in response to LPSN and nitrogen external supply, we proposed that the DNA MTase could be differentially regulated. Greco et al. (2019) reported that cadmium and copper both could induce the upregulation of the DNA methyltransferases seagrass . Villalos stress . We founll sites , 56 and ll sites . DRM3 isll sites . CMT3 isll sites , 59, 60.ll sites , 61. DNAll sites , 63.Epigenetic regulation is mediated by differences in DNA methylation at cytosine residues and by post-translational histone modifications , and hisPrevious studies report the inheritance of environmentally induced epigenetic variations , which cOsNAR2.1 in both WYG7 and Ox1 lines could not be influenced. Therefore, the phenotype of both wild type and high N-use efficiency Ox1 lines was strongly influenced by the external N deficiency, overcoming the influence of parent seed N content. In the HN field, the parent seed nitrogen content decreases induced DNA methylation changes at the epigenetic level and significantly decreased the expression of OsNAR2.1, resulting in a heritable phenotype of N deficiency over two generations in the Ox1 line.We hypothesize that in the N deficiency field, LPSN caused a significant number of gene methylation changes, but the expression of OsNAR2.1 in LPSN filial Ox1 was significantly reduced compared to HPSN filial plants in high external N, but the expression of OsNAR2.1 in LPSN filial wild type did not change in high or no external N. Therefore, we concluded that the parent seed nitrogen content decreases induced DNA methylation changes at the epigenetic level and significantly decreased the expression of OsNAR2.1, resulting in a heritable phenotype of N deficiency over two generations in the overexpression line. Our results maybe point out an innovative view in plant growth responds to endogenous nitrogen stress and indicate a new direction in breeding of high nutrient efficiency plants.In our study, we found that the parent seed N content decrease in Ox1 could retard filial seedling growth. Furthermore, induce a decrease in filial seed N content, plant N content, and grain yield in mature with high external N supply. However, the growth of WYG7 in both seedling and mature are not influenced by LPSN. To determine the mechanism underlying the phenotype, we sequenced the methylation data of Ox1 and WYG7 lines with different parent seed N content in different external N. The results demonstrated that LPSN caused massive gene methylation changes and differential methylation gene enriched in over 20 GO pathways in filial Ox1. Furthermore, the expression of OsNAR2.1 overexpression line (Ox1) with background of Oryza sativa L. ssp. japonica cv. Wuyugeng7 (WYG7) has been described in Chen et al. (2017) [OsNAR2.1 overexpression line (Ov199) and RNAi line (RNAi) with background Oryza sativa L. ssp. japonica cv. Nippobare (NP) has been described in Yan et al. (2011) [. (2017) . OsNAR2.. (2011)  and Fan . (2011) . For pla. (2011) , 68. We v/v) hydrogen peroxide solution for 30 min, thoroughly rinsed, and washed six times with deionized water. Seeds of uniform size were germinated on the top cover of a 1 L pot with 40 holes per top cover and one seed per hole. Plants were grown in the greenhouse under natural light at day/night temperatures of 30 °C/22 °C and 60% relative humidity. The containers were filled with water with with 2.5 mM of NH4NO3 or pure water. Ten-day-old seedlings were transplanted into holes on the top cover of a 7 L pot.For plants grown in seedling with hydroponics, all seeds collected from T5 were surface-sterilized with 10%  in the same sample and presented as 2-△CT. All primers used for RT-qPCR are listed in Supplementary Table Total RNA was extracted from flag leaf tissue using TRIzol reagent . DNase I-treated total RNAs were subjected to reverse transcription (RT) with HiScript II Q Select RT SuperMix for qPCR kit  according to the manufacturer’s instructions. Quantitative assays were performed using AceQTM qPCR SYBR Green Master Mix . The relative expression level was normalized to the amount of Genomic DNA was extracted from leaves of mature plants and shipped to the Anoroad Genome Company (Beijing) for Whole Genome Bisulfite Sequencing (WGBS) library preparation and sequencing.The raw reads were filtered and trimmed to obtain clean reads and the available data were compared with the reference genome of Oryza_sativa Japonica IRGSP-1.0 to obtain the alignment results using Bismark (v0.9.0) . For eachttp://fame.edbc.org/smart /readme.html) with: (1) the read coverage of each C > 4; (2) The total number of C in each DMR > = 5; (3) The length of DMR > = 100 bp; (4) The difference of methylation level between groups was greater than or equal to 0.3; (5) The CPG site with p-value less than 1e-10 was reserved as the final DMR. Based on the results of DMR genome annotation, genes with overlapping promoter (1Kb upstream) and gene body region consider as differential methylation gene (DMG).The differential analysis of methylation was performed at the region-based level, differentially methylated regions (DMRs) . Aberranhttp://systemsbiology.cau.edu.cn/agriGOv2/index.php).GO analysis was performed using AgriGO .The data were analyzed by Duncan one-way analysis of variance (ANOVA). Statistically significant differences at Additional file 1 Table S1 Comparison of agronomic traits of WYG7 and Ox1 in +N and -N fields in S0 generation.. Table S2 Comparison of agronomic traits of WYG7 and Ox1 in S1 and S2 generation.. Table S3 Agronomic traits of WYG7 and Ox1 lines with different parent seed N content in the different N field. Table S4 Agronomic traits of WYG7 and Ox1 lines with different parent seed N content in the different N field in S2. Table S5 Samples sequencing data in this study. Table S6 Characteristics of phenotype for Ox1 and WYG7 with different seed N content for two-year different N fertilizer. Table S7 Primers for qRT-PCR. Fig. S1 Parent seed nitrogen content decrease influence on the expression of OsNRTs genes. Fig. S2 The decrease in the parent seed nitrogen content influence on phenotype. Fig. S3 Plant height and grain yield induce by N deficiency in S0 and S1 generation.. Fig. S4 The decrease in the parent seed nitrogen content influence on chorophyII content and seed N concentration. Fig. S5 Methylation level of Ox1 and WYG7 lines. Fig. S6 Differential methylation induces by field N deficiency in WYG7-HN and Ox1-HN. Fig. S7 Methylation status and gene expression of relative genes in HN field. Fig. S8 Methylation status and gene expression of relative genes in NN field. Fig. S9 Methylation status of OsNRT family genes in different N field. Fig. S10 The decrease in parent seed nitrogen content of Ov199 leads to plant height and grain yield per plant decrease. Fig. S11 The decrease in parent seed nitrogen content of Ov199 leads to total N content of seeds and plant decrease. Fig. S12 The decrease in parent seed nitrogen content of Ov199 leads to relative expression of OsNAR2.1 decrease. The decrease in parent seed nitrogen content of Ov199 leads to relative expression of OsNAR2.1 decrease. (A-D) Relative expression of OsNAR2.1 of different lines in the field with HN, MN, LN and NN fertilizer. Error bars: SD (n = 4). Significant differences between different lines are indicated by different letters .