Here, we showed that white adipose tissue (WAT) of humanized knock-in BDNFVal66Met (BDNFMet/Met) mice is characterized by an altered morphology and an enhanced inflammatory profile compared to wild-type BDNFVal/Val. Four weeks of voluntary PE restored the adipocyte size distribution, counteracted the inflammatory profile of adipose tissue, and prevented the prothrombotic phenotype displayed, per se, by BDNFMet/Met mice. C3H10T1/2 cells treated with the Pro-BDNFMet peptide well recapitulated the gene alterations observed in BDNFMet/Met WAT mice. In conclusion, these data indicate the strong impact of lifestyle, in particular of the beneficial effect of PE, on the management of arterial thrombosis and inflammation associated with obesity in relation to the specific BDNF Val66Met mutation.Adipose tissue accumulation is an independent and modifiable risk factor for cardiovascular disease (CVD). The recent CVD European Guidelines strongly recommend regular physical exercise (PE) as a management strategy for prevention and treatment of CVD associated with metabolic disorders and obesity. Although mutations as well as common genetic variants, including the  Despite the huge growth in knowledge and advances in the prevention and treatment of cardiovascular disease (CVD), this pathology is still the leading cause of morbidity and mortality in the world and is predicted to reach 23.3 million by 2030 . It is wInternational guidelines, including 2016 European Guidelines on CVD prevention in clinical practice , stronglbrain-derived neurotrophic factor (BDNF) is included among these genes. Genome-wide association studies (GWAS) have shown a strong association between the BDNF locus and anorexia nervosa, bulimia nervosa [BDNF plays an important role in energy metabolism food intake and weight control [Starting from the discovery that several rare forms of obesity, called monogenic obesity, result from a mutation in single genes primarily located in the leptin–melanocortin pathway ,16, rece nervosa , or obes nervosa ,21. Inde control ,23.BDNF Val66Met variant through reduction of the activity-dependent secretion and signaling of mature BDNF, is associated not only to neuro-psychiatric disorders [BDNF Val66Met polymorphism has a significantly higher body weight than wild-type littermates [In this context, the common human isorders  and CVD isorders  but alsoisorders ,28,29,30termates , associatermates . The fretermates ,33. In ttermates .BDNF Val66Met polymorphism, obesity, and thrombosis, by analyzing the adipose tissue profile in BDNFMet/Met mice, and to evaluate the ability of PE to affect adipose tissue and reduce the prothrombotic phenotype in BDNF Val66Met knock-in mice. Finally, in vitro studies were performed to investigate the functional relevance of BDNF Val66Met polymorphism on adipogenesis.The aim of the present study was to investigate the relationship between the Val/Val and BDNFMet/Met littermate mice, generated by Chen Z-Y et al. [All experiments were performed in adult (3–4 months old) wild-type BDNFY et al. . Only maVal/Val and BDNFMet/Met mice were weighed and allocated randomly into running (RUN) and control  groups in cages equipped with or without running wheels, respectively, for 4 weeks with free access to food and water. Four sedentary control mice were housed in a standard polypropylene mice cage. Four runner mice were housed in standard polypropylene rat cages, with free access to two running wheels (12 cm diameter and 5.5 cm width). The greater dimensions of cages for runner mice were necessary for an adequate setup of running wheels. Running wheels were connected to an electronic counter, and the total distance ran was recorded daily. The average distance ran by a single mouse was calculated by dividing by 2 the total distance recorded per wheel (two running wheels × cage × four mice). The average distance ran by a single mouse, in our model, was comparable with the average distance reported by others [Mice underwent voluntary PE protocol as previously described ,36. Briey others ,36,37,383  was applied for 3 min, and the flow was recorded for 30 min. An occlusion was considered to be total and stable when the flow was reduced by >90% from baseline until the 30 min observation time.Experimental arterial thrombosis was induced as previously described . BrieflyBlood was collected by cardiac venipuncture into 3.8% sodium citrate (1:10 vol:vol) from anesthetized mice, and white blood cells and platelets were counted optically.+ gate.Platelet/leukocyte aggregates were analyzed as previously described . Briefly2/95% air atmosphere. Cells were plated in 6-well plates at a concentration of 3.5 × 104 cells/mL, and when they reached 80% confluence (day –2), they were treated with 10 ng/mL of ProBDNFVal or ProBDNFMet synthetic peptide  [BDNF expression occurring in physiological conditions during adipogenesis [The C3H10T1/2 cell line has been used to evaluate the effect of different compounds on adipogenesis processes, as previously shown ,42,43. C Israel) ,45,46 toogenesis . Forty-eAfter ProBDNFVal or ProBDNFMet treatment, C3H10T1/2 cells were analyzed during adipogenesis by flow cytometry, as previously described . BrieflyOil-Red-O staining was performed as already described  on day 9Total RNA was isolated from mouse adipose tissue or C3H10T1/2 cells with TRIzol Reagent  and a Direct-zol RNA extraction kit  according to the manufacturer’s instructions. One microgram of RNA was reverse-transcribed using an iScript Advanced cDNA Synthesis Kit .® Universal qPCR mix containing the specific primers and fluorescent dye SYBR Green . RT-qPCR was carried out in triplicate for each sample on the CFX Connect real-time System  as previously described [BDNF pathway were assessed .Samples of cDNA were incubated in 15 µL Lunaescribed . Gene exImmunocytochemistry and the analysis of adipocytes were performed in inguinal (ingWAT) and epididymal (epiWAT) white adipose tissue. Tissues were fixed overnight in 4% formalin, embedded in paraffin, cut at 5 μm, and mounted on polarized slides . Five seThe number and size of adipocytes were evaluated in hematoxylin and eosin stained sections by counting five 5× microscopic fields for each tissue sample using the ImageJ-Macro Adipocytes Tool. Images were taken with a Zeiss Axioskop  equipped with an intensified charge-coupled device (CCD) camera system .p < 0.05 were considered statistically significant. Data are expressed as mean ± SEM.Statistical analyses were performed with GraphPad Prism 7.0 and SAS versus 9.4 software . Data were analyzed by Student’s t-test, two-way or three-way ANOVA with or without repeated measures for main effects of genotype and treatment or time and stimuli, as reported in every graph, followed by a Bonferroni post hoc analysis as appropriate. When data distribution was not normal, the variables were included in the analyses after logarithmic transformation. Values of Met/Met mice have a significantly greater body weight compared to wild-type BDNFVal/Val littermates  stages of differentiation showed that pretreatment with the peptide carrying the Met mutation determined a significant down-regulation of adipogenic genes, including A levels A. In addA levels . HoweverA levels C.Met/Met mice, only Sorl1 was enhanced by ProBDNFMet treatment at late stages of differentiation (day 9)  .BDNF Val66Met knock-in mice.According to international cardiovascular guidelines  that recVal/Val and BDNFMet/Met mice underwent 4 weeks of voluntary PE in cages equipped with a running wheel. As previously reported [Val/Val and BDNFMet/Met mice  in our experimental setting. In addition, we showed that PE did not affect the percentage of ingWAT and epiWAT on the total body weight in both BDNFVal/Val and BDNFMet/Met mice, compared to sedentary mice, whereas the morphology of adipose depots was modified as provided by histological analyses  and of the mediators of inflammation such as Pai-1, Tnf-alpha, and Il-6, which is in line with the well-established paradigm that overweight and obesity are related to adipose tissue inflammation [Adipoq measured in the epiWAT of BDNFMet/Met mice and the higher number of circulating leukocytes and platelets as well as their activation state, might well summarize the relationship between adipose tissue inflammation and thrombosis. Indeed, the inflammatory profile of adipose tissue in obese subjects as well as the increased presence of these proteins in the circulation have a direct role in the onset and progression of the pathology [The adipose tissue accumulation found in BDNFammation . In addiathology ,69,70, eathology ,72,73,74athology .Pparγ along with those of adiponectin found in BDNF mutant mice might also contribute to the observed adipose tissue inflammation. It is well known that PPARγ, alongside the role of master regulator of adipogenesis, is also involved in the regulation of adipose tissue inflammation. In particular, it was demonstrated that PPARγ downregulates inflammatory adipokines in WAT. Specifically, PPARγ activation downregulates the expression of inflammatory markers such as MCP-1 and TNFα and, thus, reduces inflammation in activated macrophages [PPARγ activation induces adiponectin expression, thus further contributing to the reduction of chronic inflammation [Of note, the reduced levels of rophages ,76,77,78ammation .BDNF expression was markedly greater in epiWAT of mutant mice, supporting our hypothesis that the BDNF Val66Met polymorphism contributes to adipose tissue pathophysiology.Remarkably, BDNF-(si)RNA-mediated knockdown in the 3T3 cell line showed a reduced adipogenic differentiation ability, supporting the hypothesis that BDNF expression is of functional relevance for adipogenesis. In addition, it was reported that BDNF expression is dramatically downregulated during adipocyte differentiation, and mature adipocytes only marginally contribute to the production of BDNF in the adipose tissue [Indeed, studies performed using e tissue .Pparγ and upregulated Sorl1 expression, and it increased the percentage of mature adipocytes evaluated in the flow cytometry analysis, suggesting a direct role of the BDNF Val66Met polymorphism in the regulation of adipogenesis. However, Pro-BDNFMet was not able to affect Adipoq and Adra2a as well as Pai-1 expression, leading us to hypothesize a more complex process that may involve the fraction stromal vascular cells. Indeed, it is suggested that mesenchymal progenitor/stem cells, preadipocytes, endothelial cells, pericytes, T cells, and macrophages, and not mature adipocytes, are the main source of adipokines and PAI-1 in adipose tissue. Of note, the stromal vascular fraction in adipose tissue increases with an increasing degree of obesity [Interestingly, we showed that the treatment of C3H10T1/2 cells with Pro-BDNFMet before cell commitment well recapitulated the expression profile of genes that were found altered in the epiWAT of mutant mice. Pro-BDNFMet reduced  obesity .Adipose tissue accumulation represents an independent and modifiable risk factor for CVD , and regMet/Met mice, four weeks of PE was sufficient to change epiWAT morphology and the inflammatory profile with a concomitant reversion of the prothrombotic phenotype. In particular, the change in adipose tissue morphology observed in BDNFMet/Met running mice was accompanied with a reduction in Sorl1 and Adra2a expression, thus suggesting that PE might improve the metabolic profile of mutant mice, ultimately affecting lipolysis [In the present study, we provide evidence that, in mutant BDNFipolysis ,83,84.The beneficial effect of PE has been provided in animal studies and human trials, showing an impact on both systemic ,85 and sMet/Met mice reduced the levels of inflammation mediators, induced a switch in macrophage polarization, and decreased the number of circulating leukocytes and platelets, modifications that, in turn, occur to improve the prothrombotic phenotype observed in mutant mice. Interestingly, for the first time, we provide evidence that PE influenced differently the expression of BDNF in the two genotypes, increasing and decreasing its levels in BDNFVal/Val and BDNFMet/Met, respectively. These results might be related to the intrinsic adipose tissue morphology of BDNFVal/Val and BDNFMet/Met mice, suggesting a strong relationship between adipocyte dimension and BDNF levels. In fact, the great number of small adipocytes was associated with high levels of BDNF , and conversely, low levels of transcript were measured in epiWAT when the mean adipocyte dimension was higher . The different involvement of the stromal vascular cell fraction in sustaining the adipocyte turnover, as well as the potential contribution of the peripheral nervous system, might explain the different mRNA levels of BDNF detected in our experimental setting [BDNF transcripts in the central nervous system of mutant mice [BDNF Val66Met polymorphism impairs the beneficial neurobiological changes induced by physical exercise in mice [In line with this evidence, we showed that PE in BDNF setting ,93,94. Iant mice  might ha in mice .Cardiovascular disease still represents the first cause of mortality worldwide, and obesity is a well-known modifiable risk factor for this pathology. Of note, PE is highly recommended to manage the prevention and treatment of CVD and obesity, showing beneficial cardiometabolic effects.BDNF Val66Met polymorphism is known to be related to adipose tissue accumulation and cardiovascular risk.In human subjects, the Met/Met WAT mice.Interestingly, our in vitro data well support the role of Pro-BDNFMet in adipogenesis, in line with data obtained in the BDNFBDNF Val66Met polymorphism, we showed that 4 weeks of voluntary physical exercise was sufficient to induce positive morphological changes and reduce the inflammatory profile of the adipose tissue.Taking advantage of a mouse model carrying the human These beneficial effects might be the bases of the observed reduction in the prothrombotic phenotype detected in this animal model. Future studies are required to assess this relationship.These data indicate the strong impact of lifestyle, in particular the beneficial effect of PE, on the management of arterial thrombosis and obesity-associated inflammation in relation to genetic mutations that predisposes one, per se, to these pathologies. Nevertheless, human studies need to support these results.


Little is known about patient satisfaction, bias, stigma, and the effects of psychotherapy within the Kingdom of Jordan or the Arab world in general. The purpose of this study was to explore the perceptions of both the Jordanian host and refugee community members from the psychotherapeutic services offered at various mental health care settings in Jordan. A sample of 100 patients who received psychosocial expert interventions was recruited between October and December 2017 in Amman, Jordan. Participants were either from the host or Syrian refugee community or contacted through multiple organizations working in the mental health field. The Patient Satisfaction Questionnaire, which consists of four subscales covering 1) patient satisfaction, 2) bias toward therapy, 3) effects of therapy, and 4) stigma, was administered. As a means of investigation and exploration, descriptive statistics of participant responses are displayed. Results revealed overall high rates of satisfaction with provided services and perceived benefits of psychotherapeutic interventions. However, respondents showed ambivalence regarding bias and stigma. Subsample analyses indicated no significant differences between both communities. These findings give an understanding of perceptions surrounding psychotherapy in Jordan and some insights on therapeutic processes that may be useful for clinical applications and future research. In the last decade, political turmoil and unstable social climates have spread across many countries in the Arab region –3 . The In that context, the Kingdom of Jordan is a relatively small, middle-income country located in the Middle East with a population of nearly 10 million inhabitants , 8. SimiHigh prevalence rates contrast with only a small number of mental health institutions. In the whole of Jordan, there are only three mental health hospitals for adults, one specialized psychiatric hospital for children , 13, andThese numbers give insight into the current state of affairs regarding mental health care in Jordan, where the population size has significantly grown between the years 2004 and 2015 , 8. In aA lack of psychiatric facilities, culturally sensitive treatment programs, and professional training and poor coordination between clinicians and centralized treatment services in metropolitan cities , 5, coupWithin the Arab culture, there is a strong emphasis on the collective wellness of the group over that of the individual. For this reason, a strong attachment to social norms and values can be readily observed , 10. BecDespite growing challenges, efforts have been made to address this wide treatment gap, although resources remain scarce and national legislation supporting the beneficiaries of treatment is insufficient –3, 5 . TIn total, a sample of 100 patients receiving psychotherapeutic expert interventions were recruited between October and December 2017 in Amman, Jordan. Paper-and-pencil interviewing (PAPI) took place, whereby a trained international or local interviewer read the question to the participants and filled out the answers into the questionnaire in Arabic. Before the beginning of data collection, all interviewers received in-depth training to ensure consistency in the interview process.Inclusion criteria for the current research study were defined as the following: 1) individuals aged between 18 and 75 years; 2) belonging either to the Jordanian host or Syrian refugee community; 3) are currently or have been receiving psychotherapy or counseling sessions at organizations, which provide mental health services; 4) have attended a minimum of five sessions before the initial interview; 5) additional psychotropic treatment was allowed.Grounded in this selection process, recruitment of a randomized sample was not viable. However, within the current study design, a balance between different patient groups with regard to sociodemographic variables, such as gender and background (host and Syrian refugee community), was established.via telephone by local staff of Jiyan Foundation and CH4S. Recruitment was conducted throughout the following mental health service providers in Jordan: Al Hashmi Clinic, Bright Future, Caritas Jordan, CharitéHelp4Syria, Center for Victims of Torture, Institute for Family Health, Nippon International Cooperation for Community Development, Happiness Again, Saudi Hospital, and Médicins du Monde. A signed informed consent was requested from all patients who met the inclusion criteria before officially joining the study.As a sampling method, cooperating local organizations contacted possible study participants directly and asked for their willingness in participating. Next, suitable patients who gave their consent were contacted Because of logistic challenges, a matching of gender between interviewers and participants could not always be ensured. All participants were reimbursed for their travel costs. Furthermore, all respondents received a CH4S telephone hotline number in case follow-up psychological support was needed. After completion of the pencil-and-paper questionnaires, data were translated into the English language by local translators and entered into a spreadsheet using the Statistical Package for the Social Sciences (SPSS) 25, iOS. The study design has been approved by the ethical committees of Charité-Universitätsmedizin, Berlin, Germany, the Department of Health (DoH), and the Ministry of Health (MoH), Amman, Jordan.Because of the scarcity of appropriate and culturally sensitive questionnaires, which measure patient perceptions and preferences regarding psychotherapy in the Arab world, a comprehensive assessment tool was developed in close collaboration with the NGO Misereor for this research project. The Patient Satisfaction Questionnaire (PSQ) was designed with an aim to assess patient perspectives on the process of psychotherapy, including beliefs, perceptions, and expectations regarding patient satisfaction, perceived bias, stigma, and the effects of therapy in Arabic-speaking countries.During the development of the questionnaire, regular feedback concerning the acceptability, feasibility, and comprehensibility was collected through pilot trials, enabling continuous optimization until saturation was reached.–The PSQ is a 26-item brief self-report assessment tool consisting of four subscales, which cover the core domains of mental health care delivery: (1) therapeutic relationship, (2) bias, (3) effects of therapy, and (4) stigmatization  25, MacOS-X. The central tendency of continuous measures was represented using frequencies, percentages, means, and the variability with standard deviations as well as the range of each variable. All categorical variables were represented with percentages along with the actual counts so that missing measures are apparent in For the current study, 100 participants, 39 males and 61 females, were recruited, ranging in age from 18 to 74 years. A vast majority of 98% stated Arab as their ethnicity and indicated Islam as their religious affiliation. Two-thirds of the sample were from the Syrian refugee community (65%) whereas one-third was of Jordan origin (35%). USatisfaction = –1.13, p = 0.26, tBias(96) = –1.576, p = 0.12, UEffectsofTherapy = –1.07, p = 0.27 and UStigma = –1.62, p = 0.11. Therefore, the current results present total responses of participants.Overall, descriptive results of frequencies concerning each of the four subscales are displayed in Participants rated their satisfaction regarding the consultation and treatment of the therapist on eight items. Overall, results showed high satisfaction rates on the subscale, with an average mean of 4.71 (0.59). In total, 77.8% rated the interpersonal level of treatment as “very good” and 16.3% as “good.” Only 0.2% described experienced treatment delivery as “very poor” and 0.4% as “poor,” whereas 5.2% rated it “fair.” Attitudes in terms of bias toward the therapists were rated on a subscale with six items. In total, bias against the therapist was relatively low; instead, the average response of 3.54 (1.14) indicates high rates of acceptability. With 29.4% stating “totally agree” and 22.7% “somewhat agree,” a majority of 52.1% accepted possible differences in gender, religious affiliation, ethnicity, or political attitudes with their therapist. Still, 10.4% expressed their disapproval with “totally disagree” and 7.2% with “somewhat disagree,” indicating minimal rates of bias against their therapist, whereas 30.3% remained “undecided.” Concerning the effects of therapy, the overall mean of participants displays overall high rates of approval, with 4.44 (0.80SD) on average. In total, 60% of the respondents stated “totally agree,” whereas 28.8% endorsed the beneficial effects with “somewhat agree,” resulting in 88.8% of respondents who expressed positive effects of the therapy. Merely 3.1% believed that the therapy led to no favorable effects, with rates at 0.8% for “totally disagree” and 2.3% for “somewhat disagree,” respectively, whereas 8.0% were undecided. Because all four items on the stigma subscales were phrased with reversed items, lower rates of approval indicate lower degrees of stigma. A total mean score of 2.45 (1.44SD) demonstrates moderate rates of self-stigma. However, the mean value does not reflect respondents’ neutrality; instead, many participants either indicated self-stigma with 20.9% for “somewhat agree” and even 13.6% for “totally agree” or rejecting self-stigma with 40.7% stating “totally disagree” and 21.1% “somewhat disagree,” whereas only 3.7% responded “undecided.” Answer patterns across all four items and average scores for the stigma subscale are shown in To our knowledge, this is the first exploratory study that has been conducted in Jordan investigating both refugee and host community perceptions regarding patient satisfaction, bias, stigma, and the effects of psychotherapy. Main results showed i) a high level of satisfaction with the treatment, ii) a high level of acceptance toward the therapist, iii) an overall positive evaluation of the treatment process, and finally iv) a moderate level of self-stigmatization. Because knowledge and information are scarce regarding the attitudes and opinions of Arabs and refugees on the process of psychotherapy, this study offers the first insights from the perspective of beneficiaries who have had contact with mental health services in Jordan.Understanding patient satisfaction and the effects of therapy are important indicators for health care quality . PerspecAs previously mentioned, there was a high level of overall satisfaction reported in our study. Furthermore, the majority of the participants in the current sample (61%) ranged between 18 and 39 years. An interesting study reported the highest level of satisfaction to be observed in the age group of 18 to 34 , whereasIn 1975, Luborsky, Singer, and Luborsky investigated the hypothesis that regardless of the type and method of psychotherapy, across different treatments, only small differences in effect sizes were observed when comparing active treatments . Since tOverall, there were no major biases toward psychotherapists observed in our study. In general, participants seemed to have no reservations regarding their therapists’ religious orientation, country of origin, and ethnic background. Only two biases appeared worthy of further investigation, one being gender related and the other concerning a difference in opinion about their national politics. Some research in this field hypothesizes that, within the Arab culture, there is a clear preference to and acceptability of females as therapists , 41. At Regarding the bias of differences in political opinion between therapist and client, it seems like a general pattern emerged, ranging between undecided and disapproval. The ambivalence in their response may be because participants are mostly individuals who are fleeing from war and others from the host community who are impoverished , 9. OngoAcross the literature, various social barriers have been identified in the access and delivery of mental health services , 17, 18.Finally, regarding the item on family planning, there seemed to be a clear split in the responses toward this item. Although almost half of the cohort (47.5%) disagreed to the possible disadvantages concerning family planning and family life because of psychological problems, the other half (51.5%) agreed to it posing as a barrier. Family reputation is an integral feature for choosing a marriage partner within the Arab culture . Among sThis exploratory study gives a detailed insight into the perception of psychotherapeutic services in a large cohort of host and refugee beneficiaries in Jordan. Although samples were collected from a diverse number of hospitals and organizations, a potential selection bias in this convenience sample may have occurred during recruitment because more compliant and more open respondents may have agreed to take part in the study. This may also have affected general positive responses throughout the questionnaire and overall positive ratings. This sample was not randomized; however, the study design attempted to create a balance between host and refugee community. Furthermore, several methodological limitations were also present in this research project, namely, that no data were available on psychiatric diagnosis, current psychopathology, or total length of treatment. It would also be ideal if future research could focus more on collecting data from qualitative interviews. This approach would allow for rich and in-depth further investigations that were not within the scope of this study.In conclusion, although the preliminary results should be interpreted with caution, the present study provides evidence toward a positive attitude toward psychotherapists and psychotherapeutic treatment in both the refugee and host communities in Jordan. These findings validate the argument that, in an environment where psychiatric disorders are usually stigmatized, contact to mental health services not only improves individual well-being but also positively alters attitude, at least in those receiving professional support. This knowledge may not only be useful in the case of Jordan but also be applied in other Arab countries with comparable mental health care structures. Further studies should be conducted with a more rigorous population selection, sample size, intervention description, and possibly a mixed-methods approach.The datasets generated for this study are available on request to the corresponding author.The study was carried out in accordance with the recommendations of the Ministry of Health of the Hashemite Kingdom of Jordan with written informed consent from all subjects. All subjects gave written informed consent in accordance with the Declaration of Helsinki. The protocol was approved by the Ministry of Health in the Hashemite Kingdom of Jordan. The original ethics approval is available and can be submitted upon request.CK contributed to the interpretation of data, writing (sections: Introduction and Discussion), and revision. KB contributed to the writing and data analysis (sections: Methods and Results). EH contributed to the conceptualization of study design, revision, and supervision. SS contributed to the data collection and conceptualization of study design and logistics. JS contributed to the data collection and conceptualization of study design. MB contributed to the conceptualization of idea, study design, revision, and supervision. All authors read and approved the final manuscript.The study was funded by the German Ministry of Economic Cooperation and Development within the Special Initiative “Psychosocial Support for Syrian and Iraqi Refugees and Internally Displaced People.”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.


Iron deficiency (ID) in pregnancy is a common problem that can compromise both maternal and fetal health. Although daily iron supplementation is a simple and effective means of treating ID in pregnancy, ID and ID anemia (IDA) often go unrecognized and untreated due to lack of knowledge of their implications and competing clinical priorities.p < 0.0001). In addition, a significantly lower proportion of women received an RBC transfusion during their pregnancy  or immediately following delivery and in the 8 weeks following . Limitations of this study include the use of aggregate data extracted from the EPR, and lack of a control group.In order to enhance screening and management of ID and IDA in pregnancy, we developed a novel quality-improvement toolkit: ID in pregnancy with maternal iron optimization (IRON MOM), implemented at St. Michael’s Hospital in Toronto, Canada. It included clinical pathways for diagnosis and management, educational resources for clinicians and patients, templated laboratory requisitions, and standardized oral iron prescriptions. To assess the impact of IRON MOM, we retrospectively extracted laboratory data of all women seen in both the obstetrics clinic and the inpatient delivery ward settings from the electronic patient record (EPR) to compare measures pre- and post-implementation of the toolkit: a process measure of the rates of ferritin testing, and outcome measures of the proportion of women with an antenatal (predelivery) hemoglobin value below 100 g/L (anemia), the proportion of women who received a red blood cell (RBC) transfusion during pregnancy, and the proportion of women who received an RBC transfusion immediately following delivery or in the 8-week postpartum period. The pre-intervention period was from January 2012 to December 2016, and the post-intervention period was from January 2017 to December 2017. From the EPR, 1,292 and 2,400 ferritin tests and 16,603 and 3,282 antenatal hemoglobin results were extracted pre- and post-intervention, respectively. One year after implementation of IRON MOM, we found a 10-fold increase in the rate of ferritin testing in the obstetric clinics at our hospital and a lower risk of antenatal hemoglobin values below 100 g/L  13.0%–14.11%]; post-intervention 10.6% [95% CI 9.6%–11.7%], The introduction of a standardized toolkit including diagnostic and management pathways as well as other aids increased ferritin testing and decreased the incidence of anemia among women presenting for delivery at our site. This strategy also resulted in reduced proportions of women receiving RBC transfusion during pregnancy and in the first 8 weeks postpartum. The IRON MOM toolkit is a low-tech strategy that could be easily scaled to other settings. Michelle Sholzberg and colleagues report on the implementation of a toolkit aimed at improving management of anemia in pregnancy. Iron deficiency is a common problem in pregnancy, with serious health consequences including maternal anemia requiring blood transfusions, early labor, low birth weight, and long-term developmental issues in the child.If detected early, iron deficiency in pregnancy can be treated with convenient and inexpensive daily oral iron pills.Unfortunately, due to low awareness and other competing medical issues during pregnancy, iron deficiency often goes undiagnosed and untreated.We created a toolkit including treatment algorithms, educational information for doctors and patients, modified blood work requisitions, and standardized iron prescriptions in order to help obstetricians detect and treat iron deficiency.We found that after implementation of the toolkit, there was an increase in testing for iron deficiency, a decrease in maternal anemia, and a decrease in maternal blood transfusions during and after pregnancy.The implementation of the toolkit successfully helped obstetricians detect and treat iron deficiency in pregnancy.The toolkit is a low-tech strategy that could be adapted to be used in other patient groups with iron deficiency. Iron deficiency (ID) in pregnancy is a common problem that can have serious consequences. At baseline, 9% to 11% of teenage girls and women of child bearing age in developed countries have ID . UnfortuFortunately, early detection of ID and treatment with daily iron supplementation is a straightforward and effective method of addressing ID. ID and IDA can be detected using simple blood tests of hemoglobin and ferritin . GuideliOral iron supplementation is inexpensive, convenient, available in various preparations, and, most importantly, effective . In a meAlthough ID in pregnancy is simple to diagnose and treat, it often goes undiagnosed and untreated, due to low awareness of its implications and multiple competing clinical priorities in busy obstetrical clinics. An Australian group recently published a multipronged quality improvement (QI) intitative focusing on optimizing management of ID in pregnancy and improving the management of postpartum anemia . They foOur institution, St. Michael’s Hospital, is an inner city tertiary care center in Toronto, Canada, that delivers approximately 3,000 babies annually. We describe the development and implementation of a novel QI toolkit: ID in pregnancy with maternal iron optimization (IRON MOM), designed to improve early detection and management of maternal ID and IDA. The toolkit included clinical pathways, educational resources for clinicians and patients, adjusted laboratory requisitions, and standardized iron prescriptions. The toolkit was implemented to determine if priority setting and simple process changes could increase the rate of ferritin testing, decrease the proportion of women with an antenatal (predelivery) hemoglobin value below 100 g/L (anemia), and decrease RBC transfusion rates during and after pregnancy.Our institution, St. Michael’s Hospital, largely serves a demographic from lower socioeconomic backgrounds . ApproxiThe reporting of this study is in keeping with the SQUIRE 2.0 guidelines .As part of the multimodal IRON MOM QI initiative, we developed three distinct clinical pathways at each routine obstetrical clinical visit, developed educational resources, adjusted laboratory requisitions, and created standardized iron prescriptions. The toolkit was developed through multidisciplinary collaboration between Hematology, Obstetrics, Transfusion Medicine, Nursing, and Laboratory Medicine. The goal of the IRON MOM toolkit was to enhance screening and management of ID/IDA in pregnancy.The overall initiative is described visually in Educational materials were developed for obstetrical teams and patients see . For theThe laboratory requisitions used by the obstetrics team were modified to encourage ferritin testing, as the check box for ferritin was moved to a more prominent area at the top of the sheet, near the complete blood count (CBC) check box. However, ferritin was not automatically checked.Standardized iron prescription pads with all the various iron supplementations used in the initiative were provided to the obstetrics team. Although oral iron supplementation is available over the counter, having a prescription would enforce the importance of iron supplementation, serve as a reminder to the patient, and, for some women, would allow for their insurance plan to cover the cost of the drug.Plan-Do-Study-Act (PDSA) cycles were conducted to improve the IRON MOM at each iteration .The total costs of developing and implementing the IRON MOM toolkit were low. All the materials were developed by our multidisciplinary team. The total cost of printing the materials for the toolkit was approximately CAN$500 to CAN$1,000. With the implementation of the toolkit, we predicted a large increase in ferritin testing. The average cost at our institution for a ferritin test, including labor and supplies, was CAN$0.72 per test. The toolkit was streamlined to minimize the time spent on the initiative by the obstetrician per patient.To measure the influence of the toolkit at our institution, we compared pre- and post-intervention aggregate laboratory data extracted from the electronic patient record (EPR), using a time series design ,21. The We decided on one process measure and three outcome measures to compare pre- and post-intervention. Our process measure was the rate of ferritin testing in the obstetric clinics. We selected ferritin testing as our process measure, as a low ferritin is the best test for ID in pregnancy, with higher sensitivity and specificity than transferrin saturation, serum iron, or erythrocyte protoporphyrin values . Our outFrom January 1, 2012, to December 31, 2017, laboratory data of all women seen in both the obstetrics clinic and the inpatient delivery ward settings were extracted from the EPR. Data extracted include ferritin and hemoglobin (from the CBC) values. All ferritin requests at the obstetrics clinic were assumed to be ordered for purposes of ID screening. All CBC requests at the obstetrics clinic were assumed to represent a routine clinic visit. We considered the first hemoglobin measured during their hospitalization for delivery to be the antenatal hemoglobin. Women with multiple hospital admissions within a 280-day period had only the first hemoglobin measurement of their last admission for delivery included as their antenatal hemoglobin. Women with multiple pregnancies in the time periods of interest were included and were identified as having a new pregnancy by the timing of the delivery ward hemoglobin measurements being greater than 280 days apart. RBC transfusion data were obtained from the transfusion medicine laboratory and linked to women involved in the study through their medical record number.For each study month, the rate of ferritin testing in the obstetric clinic was calculated as the number of total clinic ferritin tests divided by the total number of CBC tests, multiplied by 100, to simplify interpretation. Changes in monthly ferritin rates from pre- to post-intervention were evaluated using interrupted time series analysis –25.Pre- and post-intervention distributions of antenatal hemoglobin values for all tests were initially compared using the chi-squared test when dichotomized. Considering each woman and each study period, antenatal hemoglobin values were recoded as 1 if any test value was <100 g/L or 0 if all tests yielded values ≥100 g/L. However, because women could be pregnant and provide data during both periods, we used generalized estimating equations with the binomial distribution and logit link to estimate the risk (predicted probability) of ever having values <100 g/L for pre- and post-intervention periods.Stata Statistical Software: Release 13. College Station, TX: StataCorp LP).Considering the first pregnancy during the study period, the proportion of women who received at least one RBC transfusion pre-intervention compared with post-intervention was analyzed for two time periods, during pregnancy (within 10 months of delivery), and immediately following delivery or in the 8-week postpartum period, using the chi-squared test. Data were analyzed using SAS software version 9.4  and Stata 13.0 , and as such, the Privacy Office at St. Michael’s Hospital assumes the responsibility of reviewing projects and approving the use of Personal Health Information for the specific objectives outlined in our protocol. The privacy office prohibits access to data for this project, to protect patient privacy.In total, there were 3,692 ferritin tests and 18,682 CBC tests in the obstetric clinic, and 19,885 antenatal hemoglobin tests available from the delivery ward during the entire study period. In the pre-intervention period from January 1, 2012, to December 31, 2016, there were 1,292 ferritin tests and 15,714 CBC tests from the obstetric clinic, and 16,603 antenatal hemoglobin tests from the delivery ward. In the post-intervention period from January 1, 2017, to December 31, 2017, there were 2,400 ferritin tests and 2,968 CBC tests from the obstetric clinic, and 3,282 antenatal hemoglobin tests from the delivery ward.The pre-intervention period was composed of 60 months in which the mean number of ferritin tests in the obstetric clinic per month was 21.5 (standard deviation [SD] 10.6), the mean number of CBC tests in the obstetric clinic per month was 261.9 (SD 30.5), and the mean monthly rate of ferritin testing was 8.4 per 100 tests (SD 4.4 per 100).The post-intervention period was composed of 12 months, in which the mean number of ferritin tests in the obstetric clinic per month was 200.0 (SD 25.3), the mean number of CBC tests in the obstetric clinic per month was 247.3 (SD 30.3), and the mean monthly rate of ferritin testing was 81.0 per 100 (SD 6.3 per 100).p < 0.0001). Although post-intervention rates decreased over time, there was not a significant change, as expressed by the slope estimate of −0.3 per 100  , which was statistically significant (475) see .n = 14,356 or 81.4%) were captured only in the pre-intervention period, 2,617 (14.8%) had data during the post-intervention period, while 3.8% (or 663) had data for both pre- and post-intervention periods. Using generalized estimating equations, we estimated that the risk of antenatal hemoglobin values less than 100 g/L was lower in the post-intervention period than in the pre-intervention period .A total of 17,636 unique women were represented in the 19,885 delivery ward antenatal hemoglobin tests. Most women (p = 0.0499). Immediately following delivery up to the 8-week postpartum period, 342 women out of 15,019 (2.3%) had at least one RBC transfusion pre-intervention compared with 41 out of 2,617 (1.6%) post-intervention (p = 0.0214).For this analysis, we only considered the first delivery during the study period; as such, 15,019 women were only counted once in the pre-intervention period and 2,617 women were counted in the post-intervention period. During pregnancy, 181 women out of 15,019 (1.2%) had at least one RBC transfusion pre-intervention compared with 20 out of 2,617 (0.8%) post-intervention , a minority were not, which likely attenuated the effect of the IRON MOM toolkit. Fourth, our study did not include an assessment of compliance with oral iron supplementation. Poor compliance with oral iron supplementation could also have attenuated the effect of the toolkit. Fifth, as this was a QI initiative, there was no comparator group nor randomization. Therefore, we could not control nor limit opportunities for bias.In conclusion, the IRON MOM toolkit is an innovative means to address early detection and management of ID in pregnancy. The toolkit incorporates clinical pathways at each routine obstetrical clinical visit, educational resources, adjusted laboratory requisitions, and standardized iron prescriptions. IRON MOM simplifies and streamlines ID detection and management for clinicians and enhances patient education. The toolkit is simple to implement, with a potential for high impact, and is generalizable. It could be adopted for use in other obstetric care centers or adapted to serve other populations with a high burden of ID, such as nonpregnant gynecology patients or those with gastrointestinal malignancy. Future directions for IRON MOM include the implementation of a simplified digital format that is accessible on mobile devices, and the development of a large cluster-based randomized controlled trial to definitively assess the effect of IRON MOM.S1 Fig(PDF)Click here for additional data file.S2 Fig(PDF)Click here for additional data file.S3 Fig(PDF)Click here for additional data file.S4 Fig(PDF)Click here for additional data file.S5 Fig(PDF)Click here for additional data file.S6 Fig(PDF)Click here for additional data file.S7 Fig(PDF)Click here for additional data file.


Malus asiatica Nakai., a special type of tea that is consumed in the same way as green tea. To study the effect of Wu Shan Shen Cha-derived flavonoids (WSSCF) on lesions in the stomach, a 15% hydrochloric acid–95% ethanol (volume ratio 4:6) solution was used to induce gastric injury in mice. The degree of gastric injury was assessed using tissue specimens, and the effects of WSSCF on the serum levels of antioxidant enzymes were investigated. The results showed that WSSCF could alleviate the damage of the gastric mucosa and gastric wall caused by the hydrochloric acid–ethanol solution, decrease the tissue and serum levels of malondialdehyde (MDA) in mice with gastric injury, and increase the serum levels of superoxide dismutase (SOD) and glutathione (GSH). The results of quantitative polymerase chain reaction (qPCR) showed that WSSCF could increase the mRNA expression of Mn-SOD, Cu/Zn-SOD, catalase (CAT), endothelial nitric oxide synthase (eNOS), and neuronal nitric oxide synthase (nNOS) in tissue specimens from mice with gastric injury and decrease the expression of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS). At the same time, the results of the high concentration of WSSCF (WSSCFH) group were closer to those of the drug (ranitidine) treatment group. Wu Shan Shen Cha-derived flavonoids had a good antioxidant effect, so as to play a preventive role in alcoholic gastric injury.Wu Shan Shen Cha is the leaf of  A high calcium concentration can activate phospholipase A2, promote phospholipase degradation, decrease the membrane phospholipid content, and increase the membrane permeability to Ca2+ [2+-Mg2+-ATPase, suggesting that oxygen free radicals may trigger calcium overload [Alcohol (ethanol) consumption by humans is on the rise, and the incidence of digestive tract diseases in the drinking population is significantly higher than that in the non-drinking population . In addi to Ca2+ . In addioverload . The damoverload .Malus asiatica Nakai. This tea comes from a pure wild plant that is rich in various trace elements, polyphenols, and flavonoids, which are beneficial to human health. It is a natural beverage for relieving thirst, reducing fatigue and weight, lowering blood pressure, awakening the brain, and providing spiritual nourishment [Wu Shan Shen Cha, produced in the Three Gorges Valley of the Yangtze River in China, is a type of tea refined from the leaves of the unique and local rishment . The locrishment ,8,9. Flarishment . In addirishment . Recent rishment ,13. In rIn this study, the effects of flavonoids extracted from Wu Shan Shen Cha (WSSCF) on alcohol-induced gastric injury were studied in mice, and the mechanism of action was defined. It was found that WSSCF mediated their biological effects through an anti-oxidative mechanism, which provides a theoretical basis for the application of Wu Shan Shen Cha as a traditional resource.2CO3, and 2.6 g of Na2B4O7 were added to 600 mL of a 40% (volume ratio) ethanol solution. WSSC was soaked in water at 50 °C for 30 min and centrifuged , followed by the collection of the supernatant. Flavonoids in the precipitate were extracted using the aforementioned procedure, and the supernatants from two centrifugation steps were combined. The pH was adjusted to 4.0 with hydrochloric acid, and the flavonoid extract was obtained using steam-drying ethanol rotary evaporation.Approximately 100 g of WSSC was crushed into powder, and 60 g of WSSC powder, 1.3 g of NaRutin was dissolved in 90% ethanol solution to dilute the compound to concentrations of 10, 20, 30, 40, and 50 μg/mL. The Kudingcha flavonoid extract was also dissolved in a 90% ethanol solution. According to the standard rutin solution curve, the absorbance was determined at 500 nm with a spectrophotometer, and the purity of the flavonoid extract in the WSSCF extract was calculated.Specific pathogen-free six-week-old male Kunming mice were randomly divided into normal, model, low WSSCF concentration , high WSSCF concentration , and ranitidine (50 mg/kg) groups with 10 mice in each group. After feeding for 7 days, the mice in the normal and model groups were administered 2 mL of distilled water daily for 14 days, the mice in the WSSCFL group were administered 0.2 mL of WSSCF (100 mg/kg) daily for 14 days, the mice in the WSSCFH group were administered 0.2 mL of WSSCF (200 mg/kg) daily for 14 days, and the mice in the ranitidine group were administered ranitidine (50 mg/kg) daily for 14 days. After treating for 14 days, the mice in all groups were fasted but were allowed to freely drink water. After fasting for 1 day , the mice in all groups, except those in the normal group, were administered 15% hydrochloric acid–95% ethanol (volume ratio 4:6) at 0.1 mL/10 g body weight. After treating for 30 min, the mice were euthanized. Plasma was collected from the eyeballs, and stomach specimens were harvested . The volThis study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Ethics Committee of Chongqing Collaborative Innovation Center for Functional Food (201807003B).Gastric specimens were fixed in 10% formalin for 48 h, followed by dehydration, clearing, embedment in wax, and sectioning. The sections were stained with hematoxylin and eosin and observed under a BX43 optical microscope .2. Nitrite shows purple-red under the action of p-aminobenzene sulfonic acid and methylnaphthalene amine. It has a maximum absorption peak at 550 nm. Total superoxide dismutase (T-SOD) can be calculated by determining the nitrite reduced after SOD scavenging ·O2. GSH in the sample reacts with 5,5-dithio-bis-(2-nitrobenzoic acid) (DTNB) to produce stable yellow 5-thio-2-nitrobenzoic acid (TNB) and glutathione disulfide (GSSG). The GSH content of the sample can be calculated. Under acidic and high-temperature conditions, MDA can react with thiobarbituric acid (TBA) to form red-brown trimethadione; its maximum absorption wavelength is 532 nm. Whole blood was collected from mice, stored for 1 h, and centrifuged at 4000 rpm for 10 min. The serum was collected. The serum and tissue levels of T-SOD, GSH, and MDA were determined according to the manufacturer’s instructions .Nitrite is the final product of oxidizing hydroxyl group of ·O−ΔΔCt method [Stomach specimens were homogenized. Total RNA was extracted using RNAzol , and the concentration was diluted to 1 μg/μL. Thereafter, 5 μL RNA was reverse transcribed to prepare DNA. Approximately 2 μL of DNA was combined with 1 μL of the forward primer, 1 μL of the reverse primer , and 10 t method .p-Values < 0.05 were considered significant.All experiments were performed three independent times, and the data were averaged. Statistical analysis system (SAS) 9.1 Statistical Software  was used for statistical analyses. One-way analysis of variance was used to test differences between the means of the groups. y = 903.04x + 1.5209, R2 = 0.9804, By observing the absorbance value of rutin standard at different concentrations, the standard curve  in mice of the normal group than those in mice of the other groups. The mRNA levels of Cu/Zn-SOD, Mn-SOD, and CAT were higher in mice of the WSSCF and ranitidine groups than in mice of the control group. The mRNA levels of Cu/Zn-SOD, Mn-SOD, and CAT were similar between mice of the WSSCFH and ranitidine groups.The expression of Cu/Zn-SOD, Mn-SOD, and CAT in stomach tissues of mice is shown in p < 0.05) in mice of the WSSCF group than those in mice of the model group, but higher than those in normal and ranitidine groups. By contrast, the mRNA levels of eNOS and nNOS were significantly higher (p < 0.05) in mice of the WSSCF group than those in mice of the model group, but lower than those in the normal group. The mRNA levels of COX-2, iNOS, eNOS, and nNOS were similar among mice of normal, WSSCFH, and ranitidine groups.The expression of cyclooxygenase (COX)-2, iNOS, eNOS, and nNOS in stomach tissues of mice is shown in It is generally recommended that the human body drink 12 g of tea brewed every day. According to the content of 5% flavonoids, the human body intakes 600 mg of flavonoids per day and 10 mg/kg of tea flavonoids per day. The intake of flavonoids in transformed mice was about 100 mg/kg, so we chose 100 and 200 mg/kg as the experimental concentration. Flavonoids are well absorbed by the digestive system. In addition to being mainly absorbed in the large intestine, they can also be absorbed in the stomach and small intestine and play a better role through liver metabolism. The intragastric concentration used in this study was calculated according to the recommended drinking concentration of human body, so flavonoids might have good absorption and utilization rates in mice and would not produce metabolic accumulation.Alcohol produces acetaldehyde via the catalysis of ethanol dehydrogenase, and acetaldehyde produces oxygen free radicals via the action of xanthine oxidase, resulting in gastric mucosal damage. Free radical reactions are closely related to the occurrence and development of various gastrointestinal conditions . AccordiLesions can alter the composition of gastric juice due to the excessive formation of free radicals, and secretion of gastric juice or low gastric juice pH will aggravate the degree of gastric injury . The resSeveral studies have reported a correlation between alcohol-induced gastric injury and high levels of reactive oxygen species ,21. Ther2− and protect gastric tissue. Studies have shown that alcohol causes oxidative damage and produces various free radicals. Mn-SOD and Cu/Zn-SOD can inhibit free radicals in vivo and prevent gastric injury [Copper/Zinc-SOD and Mn-SOD are two isomers of SOD in mammals, and both are free radical scavengers of SOD, except that they contain different metal ions. Cu/Zn-SOD mainly exists in the cytoplasm, whereas Mn-SOD mainly exists in the mitochondria and prokaryotic cells. Mn-SOD is abundant in visceral tissues . Among tc injury . Catalasc injury .Megatrophils synthesize and release nitric oxide (NO). Excessive NO can inhibit energy production, ribonucleoside reductase activity, and DNA replication. In particular, excessive NO production can induce cytotoxic effects, DNA oxidation, DNA double-strand breakage, cross-linking, and base modification. In gastric mucosa, NO functions as a signal transducer between immune cells and epithelial cells. It not only protects the gastric mucosa, but also promotes inflammation to damage the gastric mucosa . The reaAt present, the use of Wu Shan Shen Cha is not enough just as a tea drink. WSSCF is a kind of important active substance in Wu Shan Shen Cha. It can be made into a single functional food or used as a high-quality functional food additive to exert its biological activity on the human body. However, its industrial extraction technology and processing technology in functional food need further study.In this study, the protective effects of flavonoid extracted from Wu Shan Shen Cha (WSSCF) on alcohol-induced gastric injury in mice were evaluated. It was found that WSSCF could inhibit the secretion of gastric juice in mice with gastric injury and maintain the normal pH of gastric acid, thus alleviating the damage of the gastric mucosa. By hematoxylin and eosin staining, the degree of gastric mucosal damage in WSSCF-treated mice was alleviated compared with that in model mice. WSSCF increase the serum levels of SOD and GSH, reduced the MDA content, and protected the gastric mucosa from oxidative damage caused by free oxygen radicals, which indicates that WSSCF has good antioxidant effect in vivo. Further experiments showed that WSSCF increased the mRNA expression of eNOS and nNOS and decreased the expression of COX-2 in gastric tissues, revealing that WSSCF can exert its biological effects by functioning as an antioxidant agent. Moreover, with increasing WSSCF in the stomach, the alcohol-induced gastric injury in mice was alleviated. This shows that WSSCF has better health care effect and provides a theoretical basis for its further development and utilization.


Functional gastrointestinal disorder (FGID) patients are influenced by anxiety, depression, and low sleep quality, which reduce the quality of their life. However, epidemiological data on the quality of sleep in FGID patients were lacking. This study aims to explore the sleep quality and influencing factors of the sleep quality in FGID patients. 1200 subjects, diagnosed as FGID in one of the six class-three hospitals in Tianjin, China, from January to December 2014, were recruited. The information about demographic information, the severity of clinical symptoms, psychological status , and sleep quality  was gathered.P < 0.001). The proportion of elderly patients suffering from low sleep quality was higher than that of middle-aged and young patients (P < 0.001). The binary logistic regression analysis showed that age, education, and the severity of FGID symptom were influencing factors for poor sleep quality in FGID patients. The questionnaires from 1117 participants were collected including 920 of functional dyspepsia (FD) patients, 77 of irritable bowel disease (IBS) patients, 26 of functional constipation (FC) patients, and 94 other FGID patients. The results showed that morbidity rate for FD patients who had sleep disorders was higher than those who suffered from IBS or FC ( The issue of poor sleep quality in FGID patients in Tianjin area is prominent, and elderly patients suffer lower sleep quality than other FGID patients. Age, education, and the severity of FGID symptoms are critical influencing factors which result in a drop-in sleep quality. Functional gastrointestinal disorder (FGID) includes some symptoms of morphologic and physiological abnormalities in different parts of the digestive systems. FGID, such as functional dyspepsia (FD), functional constipation (FC), irritable bowel disease (IBS), and other symptoms, lowered the life quality of patients . PatientThe morbidity of FGID in China ranges from 5.67% to 55.24% , 4. The The present study was approved by Ethics Committee of the General Hospital of Tianjin Medical University (2013030079). All the enrolled subjects have signed the informed consent. A total of 1200 patients with FGID were recruited in six class-three hospitals  from January to December in 2014. Questionnaires were used to collect health-related information of patients. Examinations of blood, urine, and stool, liver and kidney function, blood pressure, blood sugar, thyroid function, chest radiograph, gastrointestinal endoscopy, ultrasonography, and electrocardiogram were conducted for all the subjects before the investigation. The inclusion criteria of FGID are the FGID Rome III standard, and patients aged from 18 to 65 years old [10 trained investigators conducted the interview in the follow-up clinic of the hospital using structured questionnaires (after and before investigation). Participants answered questionnaires  to reflect patients' sleep quality over one month through its seven dimensions, namely, subjective sleep quality, sleep latency , sleep duration, habitual sleep efficiency , sleep disturbances, use of sleeping medication, and daytime dysfunction . The maxP < 0.05 was regarded as statistical significance.The database was input by EpiData3.0 and analyzed by SAS 9.3. The data with normal distribution was shown as the mean ± standard deviation, and those with abnormal distribution were displayed as median and range. One-way ANOVA and rank-sum test were used to compare the difference between the groups. Ratio comparison was performed with the chi-square test, and binary logistic regression analysis was used to analyze the risk factors for sleep disorders. The current investigation recovered 1117 questionnaires, including 466 males and 649 females, with an average age of 50.8 ± 11.7. Among all subjects, there were 920 FD patients, 349 males and 571 females, with an age of 50.9 ± 11.5; 77 IBS patients, 53 males and 24 females, with an age of 49.7 ± 12.1; 24 FC patients, 18 males and 6 females, with an age of 53.0 ± 10.9; 94 other types of FGID patients, 63 males with an age of 49.8 ± 13.3. Overall, there is no significant difference among all groups.Among all the investigated FGID patients, the incidence of sleep disorder of each group varied within 71.28%–87.10%; the incidence of suffering from both a sleep disorder and abnormal psychological problems was 69.36%–83.56%, while the incidence of no sleep disorder and abnormal psychological problems was only 0–4.26%. The incidence of sleep disorder of FD patients was 87.10%; their incidence of suffering from both sleep disorder and abnormal psychological problems was 83.59%, higher than other types of FGIDs. A substantial difference between the abnormal psychological problem rations did not exist among groups .The enrolled patients were assigned to different groups according to their age. The results demonstrate that there was no statistical difference in the course of disease and gender among the young, middle-aged, and elderly groups, while there was a difference in education (with the young group having a higher ratio of well-educated patients). Age affected the sleep quality of FGID patients, as seen in the results. Compared with the young and the middle-aged, the elderly stood a higher ratio of sleep disorder and together with abnormal psychological problems, a higher overall score for sleep quality. There was no significant difference in the ratio of abnormal psychological problems among all groups, as seen in The study was conducted through binary logistic regression analysis to explore risk factors of sleep disorder in FGIDs patients as they appear at advanced ages, with low levels of education, and severity of symptoms being seen as significant risk factors when it comes to sleep disorder .Sleep disorders severely affect adults' quality of life and contribute to factors leading to the death rate , 14. ThuThe present study finds that the ratio of FGIDs  is relatively high (71.77%–87.10%), and compared with other types of FGIDs, FD patients suffer from sleep problems more easily. The result is different from that of Schurman et al. in which 283 subjects with age between 8 and 17, with the Sleep Disturbances Scale for Children (SDSC) used to evaluate sleep disorder and its incidence was shown to be 45%, with no significant difference among all FGIDs . The maiAge affects sleep quality  and the Factors influencing sleep disorders are very complex. Besides advanced age, Tang et al.  found thThis study finds that FGID patients are not only confronted with severe sleep disorders but also suffer from abnormal psychological problems, through epidemiological investigation of multicenters in Tianjin; sleep disorders in the elderly are more prominent. Age, education, and severity of symptoms can be the risk factors contributing to sleep disorder and as there are differences among baseline information among all FGIDs, large samples with more rigorous epidemiological investigation are required.


Background: The association between low levels of alcohol consumption during pregnancy and children's health remains unclear because of the difficulty in ruling out residual genetic and environmental confounding factors. In this study, using a within-family sibling fixed effects design, we sought to examine the association between low prenatal alcohol exposures (PAE) and children's overall psychosocial behavior in a Japanese cohort.Methods: We used maternal and sibling data from the Japanese Study of Stratification, Health, Income and Neighborhood 2012-2013. Households were recruited from the Tokyo metropolitan area through clustered random sampling. Children under 18 years old who have siblings  and their mothers were selected. PAE status was retrospectively measured, and classified by binominal and continuous measurements. Outcome measures of children's psychosocial behavior were assessed with the Child Behavior Checklist T-score.Results: Low PAE was significantly associated with the offspring's anxiety problems  and internalizing problems , and marginally significant with the offspring's total problem scores . There was no significant difference in PAE between boys and girls when it comes to behavioral problems.Conclusions: Low PAE was associated with children's anxiety, internalizing problems and overall problems, taking into account possible unobserved genetic and environmental confounding influences. The U.S. Centers for Disease Control, the U.S. Surgeon General, the American College of Obstetricians and Gynecologists, and the American Academy of Pediatrics uniformly advise women not to consume alcohol during pregnancy –5. HowevAlthough it is widely accepted that binge drinking during pregnancy increases the risk of fetal alcohol syndrome , 11, wheThere are several reasons why it is challenging to investigate the effects of PAE on children's behavioral problems. First, mothers tend to under report their drinking levels during pregnancy because of prevailing social norms . Second,Notably, studies that considered observed and unobserved confounding factors are rare , 18, 19.The findings from the few existing studies using the sibling fixed-effects approach , 18, 19 In this study, we examined the association between low PAE and children's overall psychosocial behavior in a Japanese cohort using the sibling fixed-effects design.koseki” registration system, a compulsory domiciliary registration system in Japan that included all residents in target area . Among the individuals invited to participate, 4,385 men and women responded (31.6% response rate), including 2,184 households with children under 18 years. By wave 2, the number of households with children increased to 2,244, and of these, 1,520 households (67.7%) agreed to participate in the follow-up survey . Written informed consent was received from all the participants in the study. We excluded children who did not have the outcome data and the prenatal mothers who drink alcohol more than 2 times per week  because we focused on low-level drinkers. Therefore, the number of children in the study was 1,933. In addition, we used only sibling data for the fixed-effects models, the total number of children who had siblings and who had outcome data was 1,600 (mean age 114.1 months ± 52.5 SD). Among them, 1,046 had two siblings, 518 had three siblings, and 36 had four siblings. If the number of siblings was more than two in one family, we compared each sibling pair separately. For example, if the number of the children were three  in one household, we compared A-B, A-C, and B-C.The J-SHINE  is an ongoing cohort study established since 2010. Details of the study have been previously described . BrieflyThe J-SHINE was conducted using computer-assisted personal interviewing (CAPI), unless the participants requested a face-to-face interview. This study was carried out in accordance with the recommendations of ethical guidelines for medical and health research involving human subjects. The study protocol was approved by the ethics committee of the Graduate School of Medicine of the University of Tokyo. All subjects provided written informed consent in accordance with the Declaration of Helsinki.The J-SHINE study asked mothers to report their drinking behavior during each pregnancy retrospectively. Response categories included: (1) 2 times or more per week, (2) 1–4 times per month, (3) rarely (but not zero), and (4) never. We excluded those who responded with “2 times or more per week” to focus on low prenatal alcohol drinking during pregnancy, and combined “1–4 times per month” and “rarely,” so that PAE was binarized into “never” vs. “ever.”n = 1316, 83.7%). The T score of each CBCL score was calculated using the standardized distribution among Japanese children and mean score represents the 50th percentile, which has been shown to have good reliability and validity  regressions controlling for all observed sibling-varying and sibling-invariant variables for the full sample, as well as for the sibling sub-sample. Next, we conducted sibling fixed-effects analysis only among the siblings , with further adjustment of correlation of 3 or more siblings comparisons from the same family. In the fixed-effects model, the effects of PAE on children's outcomes were calculated by differentiating the unobserved, sibling-invariant shared factors, such as genetic or family environmental influences. Finally, we conducted fixed-effects models stratified by child sex  as a sensitivity analysis. All analyses were performed with STATA 13.0 .n = 1,933) and the sample restricted to the siblings . In the full sample, the frequencies of prenatal alcohol “1-4 times/month,” “rarely,” and “never” were 6.1, 21.7, and 55.4%, respectively. The vast majority of pregnant women in this Japanese cohort therefore fitted into the abstention or light drinking categories.Table There was a total of 491 sibling pairs whose mother never drank during pregnancies Appendix .Table We found that low PAE during pregnancy is associated with children's anxiety, internalizing and overall problems. Our findings serve as additional evidence for the deleterious impact of maternal drinking during pregnancy on children's psychosocial behavior, and are consistent with previous animal models suggesting that even a relatively small amount of alcohol during fetal development can result in an increase in synaptic connectivity specific to the basolateral amygdala and induce a subtle anxiety-like behavior in rats .in utero  or the timing of drinking during different trimesters of pregnancy. However, there is evidence that retrospective reports of prenatal substance use can be reliable , 31. NonIn conclusion, our study provides additional evidence that even low PAE during pregnancy may adversely affect children's psychosocial behaviors, especially anxiety problems. Our findings provide further support for the current recommendation of abstinence during pregnancy, based on the notion that there is no known safe threshold of alcohol consumption during pregnancy.KI analyzed the data and drafted the paper and designed the study. IK was responsible for conception and design of the study and coauthored the paper, TF was involved in the study design, supervised the data analysis, and coauthored the paper. All authors read and approved the final manuscript.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.


In aging cells, genomic instability is now recognized as a hallmark event. Throughout life, cells encounter multiple endogenous and exogenous DNA damaging events that are mostly repaired, but inevitably DNA mutations, chromosome rearrangements, and epigenetic deregulation begins to mount. Now that people are living longer, more and more late life time is spent suffering from age-related disease, in which genomic instability plays a critical role. However, several major questions remain heavily debated, such as the following: When does aging start? How long can we live? In order to minimize the impact of genomic instability on longevity, it is important to understand when aging starts, and to ensure repair mechanisms remain optimal from the very start to the very end. In this review, the interplay between the stress and nutrient response networks, and the regulation of homeostasis and genomic stability, is discussed. Mechanisms that link these two networks are predicted to be key lifespan determinants. The Anaphase Promoting Complex (APC), a large evolutionarily conserved ubiquitin ligase, can potentially serve this need. Recent work demonstrates that the APC maintains genomic stability, mounts a stress response, and increases longevity in yeast. Furthermore, inhibition of APC activity by glucose and nutrient response factors indicates a tight link between the APC and the stress/nutrient response networks. When does the aging process begin? How long can we live? Why do we age? These questions are highly debated with no distinct, definitive answers. Does aging begin when our skin starts to wrinkle, or when our hair commences to turn grey? Or perhaps aging begins after the completion of growth . Aging hThis review will summarize the current ideas describing our thoughts on the aging process. Molecular mechanisms will then be described that facilitate cellular aging. The notion that genomic instability is the driving force leading to aging and age-related disease will be discussed. Finally, the novel concept that activation of a cell cycle regulator, the Anaphase Promoting Complex, which is required for maintenance of cell health, inhibition of cancer progression, and enhanced longevity, will be explored.Longevity depends on how long our cells remain functional, which is countered by the many insults faced by cells. Pathways that maintain cellular homeostasis are genetically controlled; thus, it clearly follows that a genetic program would be in place to control longevity. A recent review links proteostasis (a housekeeping phenomenon that controls the integrity of protein structure and function) with lifespan determination, and suggests the failure of the proteostatic network occurs early in life and marks the beginning of aging . A globaOne genetic theory of aging that is appealing to evolutionary biologists is the idea of pleiotropic antagonism. Antagonistic pleiotropy suggests that genes that are beneficial in the early years become harmful in later life ,20,21. AThe genetic model of lifespan determination is not at odds with the stochastic model, and involves a genetic program that determines the length of time that our cells and bodies can function. In the end, the more capable the cell is at damage repair, occurring through stochastic intrinsic and extrinsic events, the better the odds of surviving harsh environments and living to pass on genes to descendants. Longevity of the organism starts with the health of the cells. If cell health cannot be maintained, then health of the tissues and the animal itself will suffer. Cell type, in particular, is determined by programmed genetic and epigenetic networks. In the human body, for example, each cell harbors the same sequence of DNA, yet different cells carry out very different functions. Alterations to any of these networks can signal the end to that cell. Maintenance of the cellular equilibrium supporting tissue renewal is critical to the longevity of the organism. Over time, equilibrium and cell renewal begins to fail, leading to reduced replacement of cells lost due to attrition or senescence. Thus, the failing of the genetic system, contributing to the loss of cell equilibrium and renewal through accumulating mutations, is considered a hallmark of aging. Hallmarks of aging consist of the following attributes: genomic instability, telomere attrition, epigenetic alterations, and loss of proteostasis, leading to deregulated nutrient sensing, altered mitochondrial function, and cellular senescence ,26.SIR2 (yeast SIRT gene), SNF1 (yeast AMPK), FKH1/2 (yeast FOXO), SCH9 (yeast AKT/S6K), TOR1, and RAS2, have been shown to be evolutionarily conserved genes that respond to stress or nutrients, influencing longevity [SIR2) clearly play a significant role in promoting lifespan from single cells to humans, but do genes also drive the loss of homeostasis and the aging process? While the stress response genes drive cell health and longevity in the presence of low-level stress, an example of Hormesis [SCH9, TOR1, or RAS2, which encode nutrient response proteins [Specific genes, many of which were first described and characterized in the simple lower eukaryotic yeast system  has come to light as a potential link between the stress and nutrient sensing networks. The APC is an evolutionarily conserved large ubiquitin-protein ligase (E3) that targets proteins that inhibit mitotic entrance and exit, as well as proteins that inhibit G1 maintenance, for ubiquitin and proteasome-dependent degradation . The APCansition ,57. CDH1ansition . The APCansition ,63,64,65ansition . The APCansition ,68,69,70ansition ,72, we sansition ,74,75,76ansition . Consistansition ,64.Ac), mediated by the Asf1/Rtt109 complex [Ac, H3K79Me, and H3K56Ac [Me accumulates during mitosis [Ac and H3K9Ac are reduced during mitosis but increase as cells enter G1 [Ac is important for transcriptional activation [Ac is involved in histone deposition and DNA repair [Me is required for a variety of activities including transcriptional elongation, DNA repair, and cell cycle checkpoints [Ac, Gcn5, interacts genetically and functionally with the APC [GCN5 rescued APC defects and deletion of GCN5 in APC mutants exacerbated growth defects. Furthermore, Gcn5 is targeted by the APC for degradation at the M/G1 transition [Ac as cells exit mitosis, with APC mitotic function, is at the crux of establishing an active transcriptome for continued cell cycle progression. Furthermore, if targeted degradation of Gcn5 by the APC is conserved from yeast to humans, then this may be critical for tumor suppression and maintenance of genomic stability, as increased H3K9Ac is associated with DNA damage, genomic instability, and progression of multiple myeloma [Histone post-translational modifications are involved in cell cycle progression, particularly mitosis , and in  complex ,74. Cell H3K56Ac . H3K79Me mitosis , while Henter G1 ,116. H3Ktivation ,118, H3KA repair , while Hckpoints ,121. Thu the APC ,70. Incransition . Acetyla myeloma . Consist myeloma ,64,65 an myeloma ,124. ThuCDC20 mRNA expression is observed to be elevated in cancer cells, which is associated with a poor prognosis; CDC20 knockdown is required for mitotic arrest and inhibition of cell growth [Because of the role the APC plays in cell cycle progression, initial work focused on the inhibition of the APC as a means to block tumor growth ,126,127.l growth ,128,129.l growth ,87) and l growth ,130. BotCDC20 may be inappropriately driving cells through mitosis to promote genomic instability and cancer progression, inferring that APC inhibition will be beneficial. Regulation of CDC20 is highly coordinated , and findings from others, have noted that many APCCDH1 mitotic substrate genes and proteins are elevated in cancer cells, including CDC20 [DLGAP gene [PTTG1 gene [PTTG1 and DLGAP5 in cancer patients is differentially regulated between normal tissues and tumor tissues, across 24 different types of cancer , SecuriTG1 gene ), and GeTG1 gene , hintingTG1 gene , we obsef cancer .Cdh1 when under replication stress [Cdh1 may normally act as a barrier to genome instability [While CDC20 has been linked to cancer progression, the second APC co-activator, CDH1, has been linked to tumor suppression, with earlier work demonstrating that cells lacking CDH1 have a shortened G1 phase, accumulate DNA damage, and undergo apoptosis . CDH1 isn stress ,140, andtability . Supporttability . Thus, iAPC dysfunction and cancer development could occur in several ways. Loss of either CDC20 or CDH1 is deleterious; CDC20 deletion is lethal, while loss of CDH1 leads to genomic instability ,124. In Recently, focus has shifted towards the creation of compounds that activate the APC. To do so, SAC inhibition has been targeted. Prolonged SAC, or impaired APC activity, can lead to inappropriate mitotic progression in a process called mitotic slippage ,145. ThiAPC10 increased replicative lifespan [snf1∆ mutants were also shown to interact genetically with the CAapc5 mutant; deletion of SNF1 worsened the CAapc5 defect, whereas overexpression rescued it [APC4 and APC9 [SNF1, and that increased longevity observed in the Snf1UBA mutant depended on Fkh1 or Fkh2 [CDC20 and APCCDH1 complexes and deacetylates both CDC20 and CDH1 to turn on the APC [CLB2 transcription; overexpression of CLB2 under stress conditions is toxic [FKH1 and FKH2 are expressed during G2 to drive the expression of mitotic specific genes [FKH genes are activated by a third Forkhead protein called Hcm1, which is expressed at the G1/S boundary [ubc1∆ mutants, Hcm1 remains cytosolic, FKH1 and FKH2 transcription is reduced, and SNF1 kinase activity is decreased [Cdc20 complex [FKH1 and FKH2 in APC defective cells worsened the already short replicative and chronological lifespans [373) mimicked the null FKH1 allele, reduced chronological lifespan, and increased genomic instability [373, mediated by APCCdc20 at the onset of mitosis, is required to maintain normal lifespan and genomic stability.We have reported that the yeast APC prolongs longevity (increased expression of only lifespan ), responlifespan ,65,108  stabilized Fob1, increased rDNA instability, and abolished the accumulation of modified Fob1 species. We observed that Fob1 was specifically unstable during G1 and targeted for degradation by APCCdh1 [FOB1, like that of FKH1, rescued the lifespan defect observed in APC mutants [In addition to Fkh1, the APC also targets a second lifespan determinant, Fob1, for degradation . Fob1 inlifespan ,154, whilifespan . We iden APCCdh1 . Deletio mutants ,65. TakeCdc20 function [In order to fully maximize longevity, from the beginning to the end, coupling the stress and nutrient sensing pathways may be critical. The APC may be in a position to recognize both stress and nutrients. The APC is activated by phosphorylation to promote cell cycle progression. Using mouse fibroblast NIH/3T3 cells, it was shown that the Polo-like kinase, Plk, activates the APC by phosphorylating CDC16, CDC27, and APC1  Figure . Plk in function . Conversfunction   and PKASCH9 in yeast increases yeast replicative and chronological lifespan [FKH1 and FKH2 in either the sch9∆ or tor1∆ background eliminates the observed long life . As cells entered stationary phase, it was observed that total ubiquitinated protein decreased, as did total Sch9 protein levels [SCH9 in APC mutants suppressed the chronological lifespan and oxidative stress sensitive defects in APC mutants . We also confirmed that Sch9 turnover occurs as cells enter stationary phase, and that this is blocked in APC mutants . Taken as a whole, the published and unpublished literature supports the idea that the APC sits at the apex of the stress and nutrient-sensing pathways, controlling cell cycle progression, DNA repair, and chromosome maintenance (However, how does the nutrient sensing pathway shut down when nutrients are limited? A recent report described the turnover of the nutrient sensing kinase Sch9 in yeast . Deletiolifespan ,158, andn levels . In the n levels , supportntenance .The positioning of the APC at the intersection point of the stress and nutrient sensing pathways confers importance upon this complex, as it may have the potential to protect the cells that come together to form the zygote from the aging process. The potential for aging likely begins for an individual as soon as the germ cells responsible for them are born. For the oocyte, that means during the mother’s in utero development. It will be many years before that oocyte is fertilized; therefore, plenty of time exists for damaging side effects of cell metabolism to rear their ugly heads. It is critical that the repair mechanisms within these cells are functioning optimally. As long as the APC is at its peak function, protection against cellular damage should be high. With continued proper function of the APC through the life of the germ cells and the subsequent offspring, increased healthspan may be possible.


FCER1A, MS4A2, FCER1G, VDR, GC, CYP2R1, CYP27A1, CYP27B1, CYP24A1, RXRA, RXRB, RXRG, IL4, IL4R, IL13, and IL13RA1. The study group was composed of patients with allergic rhinitis plus asthma (AR+A), patients with hypersensitivity to beta-lactams (BLs), to NSAIDs including selective hypersensitivity (SH) and cross-reactivity (CR), and healthy controls without antecedents of atopy or adverse drug reactions. We identified 148 gene variations, 43 of which were novel. Multinomial analyses revealed that three SNPs corresponding to the genes FCER1G (rs36233990 and rs2070901), and GC (rs3733359), displayed significant associations and, therefore, were selected for a combined dataset study in a cohort of 2,476 individuals. The strongest association was found with the promoter FCER1G rs36233990 SNP that alters a transcription factor binding site. This SNP was over-represented among AR+A patients and among patients with IgE-mediated diseases, as compared with control individuals or with the rest of patients in this study. Classification models based on the above-mentioned SNPs were able to predict correct clinical group allocations in patients with DHRs, and patients with IgE-mediated DHRs. Our findings reveal gene promoter SNPs that are significant predictors of drug hypersensitivity, thus reinforcing the hypothesis of a genetic predisposition for these diseases.The prevalence of allergic diseases and drug hypersensitivity reactions (DHRs) during recent years is increasing. Both, allergic diseases and DHRs seem to be related to an interplay between environmental factors and genetic susceptibility. In recent years, a large effort in the elucidation of the genetic mechanisms involved in these disorders has been made, mostly based on case-control studies, and typically focusing on isolated SNPs. These studies provide a limited amount of information, which now can be greatly expanded by the complete coverage that Next Generation Sequencing techniques offer. In this study, we analyzed the promoters of sixteen genes related to the Vitamin D pathway and the high-affinity IgE receptor, including  The prevalence of atopy, allergic diseases, and drug hypersensitivity reactions (DHRs) is increasing worldwide. In Europe, studies have estimated a prevalence of 20–25% allergic diseases in adults, with many young people being unaware of their disease  in patients with allergic rhinitis plus asthma (AR+A), BLs hypersensitivity, selective NSAIDs hypersensitivity (SH) and cross-reactions to NSAIDs (CR), as well as in healthy control individuals. The genes included in the study were A total cohort of 2,476 individuals participated in this study. All were Caucasian Spanish individuals. These included 406 healthy controls without antecedents of atopy or adverse drug reactions, 528 patients with AR+A, 561 individuals with BLs hypersensitivity, 668 patients with NSAIDs cross-reactivity (CR), and 313 selective hypersensitivity patients (SH) which were single-NSAIDs responders. Written consent for participation was obtained for all participants. Patients were recruited at Hospitals participating in the study. All the patients who were invited to participate in the study agreed to do so. Control individuals were selected among students and staff in the University and Hospitals participating in the study. Characteristics of the study groups are summarized in To get a further analysis of the sample, we put together some of the groups of patients which share a specific characteristic. Thus, we defined three new groups of study: “DHR group,” were we included all the patients with DHR: namely, patients with hypersensitivity to BLs and NSAIDs ; “DHR-IgE Group,” which comprises selective hypersensitivity to BLs and SH; and ”IgE Mediated Group,” where we included all the IgE-mediated reactions .A subset of participants were selected for this phase. A total cohort of 175 individuals participated in this NGS analysis. These included 22 healthy controls without antecedents of atopy or adverse drug reactions, 22 patients with AR+A, 43 individuals with BLs hypersensitivity, 41 patients with NSAIDs cross-reactivity (CR), and 46 selective hypersensitivity patients (SH) which were single-NSAIDs responders. Characteristics of the participants are summarized in All patients and controls participated in this phase. Analyses were carried out by using TaqMan genotyping focused on the SNVs raised after multiple comparison analyses of the NGS phase (see the results section for further details). The SNPs were analyzed in triplicate, by using SNP TaqMan assays , and following the conditions specified by the manufacturer. Assay details are as follows: FCER1G-rs36233990, Custom TaqMan® Assay; FCER1G-rs2070901, (C__15867981_20); and GC-rs3733359, (C__25652813_40).P-values were under 0.05. The association between SNPs and traits was estimated by odds ratio (OR) with a 95% confidence interval (CI) or by Relative Risk (RR) when the variation was not found in the control group. The Relative Risk was calculated by using EpiBasic, a tool for statistical analysis of tabular information, performing a stratified analysis, using the inverse variance (1/SE2) as weigh. This tool was developed as a companion to a Danish textbook on epidemiology  for the genetic associations identified in the combined dataset model as described elsewhere  Agresti,  were perIn this phase we identified 148 variations situated in the promoter region of genes related with vitamin D and FCεRI genes. The information about the variations, their frequency in the whole sample and the Hardy-Weinberg equilibrium values is summarized in http://grch37.ensembl.org/index.html) for individuals with European descent for all the variations except for the rs4020369 SNP in the GC gene, where the described frequency for Europeans is equal to 0, but in our population it shows a MAF close to 0.040, that is in agreement with the global frequency described in 1,000 Genomes for overall individuals.Among the 148 gene variations identified, 43 were novel. Within the 105 already described SNPs, 25 have not been described or studied earlier in European individuals, although they show marginal MAF in our study (only three SNPs show MAF above 0.010). Regarding known SNPs, the frequencies are concordant with the results previously described in the 1,000 Genomes public database . It is to be noted that some SNPs with a high significance after logistic binary regression analyses (See RXRG), rs3733359 (GC), rs2070874 (IL4), rs4303288, and rs4307775 (VDR) and rs2259735 (CYP24A1), were not significant after multinomial analysis. The statistically significant variables raised after this analysis were three SNPs , as well as antecedents of atopy.One hundred and three out of the 148 variants identified were at Hardy-Weinberg equilibrium (HWE) in the overall study population see , which iyses See , such asFCER1G rs36233990 SNP was monomorphic in the control group, whereas heterozygous subjects were identified in all subgroups of patients and homozygous individuals were identified in the AR+A and CR groups. Statistically significant differences were identified for this SNP in all subgroups of patients , that is, (BLs + CR + SH). The second group of patients was composed of all patients with IgE-mediated drug hypersensitivity (D-IgE) (BLs + SH). The third group of patients was composed of all patients with IgE-mediated reactions (BLs + SH + AR+A), which were compared vs. healthy controls . The twodiseases .FCER1G rs36233990 as a good variable. The variable “Antecedents of atopy” was also selected, although this was expected because none of the control individuals had antecedents of atopy. Model 1 was made by including the three SNPs with a significant P-value and all the clinical groups separately, as compared to control subjects. Model 2 included all patients with DHR compared to control individuals, and model 3 included all patients with IgE-mediated diseases vs. control individuals. The classifications per group are shown in We built models including the three SNPs with significant associations in the combined dataset analyses phase, as well as the antecedents of atopy and gender, and the pseudo R-square values for each model (those which provide the best classification of each patient in its correct group) are shown in Genetic variation is a major cause of interindividual differences in the susceptibility to a number of disorders. In this regard, a huge number of genetic association studies related to allergic disorders and drug hypersensitivity events have been carried out. Most of these studies have a case-control design interrogating only a few polymorphisms, typically, a few SNPs located within the coding region. The use of NGS techniques allows for a complete coverage of large areas thus revealing novel SNPs or analysing SNPs that are not included in most studies. In a previous NGS study in the promoter area of the genes encoding the COX-1 and COX-2 enzymes, we identified several novel SNPs. More than 70 SNPs modified transcription factor binding sites, either by disrupting existing sequences or by creating new binding sites . We have focused on the promoter gene region due to its crucial role in transcriptional activity and expression of the gene, as it has been observed for the SNPs located in the promoter of RXRG, GC, IL4, VDR, and CYP24A1 (see the Results section), statistical significance for these SNPs was not supported after multinomial analysis, except for the GC SNP. By turn, two additional FCER1G SNPs, as well as the GC SNP, were statistically significant after multinomial analyses. It is important to note that the major findings obtained in the present study are novel, since only one of the three SNPs that remained after the multinomial analysis have been previously related with atopy or drug hypersensitivity. Among these, one of the FCER1G SNPs is novel and hence have not been studied before, and the other one has been related with food sensitization . On the other hand, the variant allele T leads the disappearance of a binding site for the transcription factor p300. Our own previous findings supported a role of FCER gene variations in patients with AR+A, but not in patients with SH . The frequencies of carriers for other two SNPs are 47 and 10.5% for FCERIG rs2070901 and GC rs3733359, respectively. Therefore, the presence of these gene variations cannot explain, by itself, the development of most cases of DHR. However, the SNPs raised in this study, point to mechanisms involved in DHR and add novel information that can be used as a proof of mechanism.In summary, our findings show that the analysis of the gene promoters is useful for the identification of genetic biomarkers of risk for DHRs and AR+A. Models using these gene variations allow a high degree of prediction, that is, correct group allocations  based onEG-M and JA contributed conception and design of the study. JG-M, CC, JC-G, NB-L, GC, ID, MB, and MT recruited and characterized patients. MM performed the statistical analysis. GA wrote the first draft of the manuscript. MM, EG-M, and JA wrote sections of the manuscript. All authors contributed to manuscript critical revision with important intellectual contribution, read, and approved the submitted 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.