This study aimed to explore the related factors and strengths of hepatic cirrhosis complicated with hepatic encephalopathy (HE) by multivariate logistic regression analysis and tabu search-based Bayesian networks (BNs), and to deduce the probability of HE in patients with cirrhosis under different conditions through BN reasoning. Multivariate logistic regression analysis indicated that electrolyte disorders, infections, poor spirits, hepatorenal syndrome, hepatic diabetes, prothrombin time, and total bilirubin are associated with HE. Inferences by BNs found that infection, electrolyte disorder and hepatorenal syndrome are closely related to HE. Those three variables are also related to each other, indicating that the occurrence of any of those three complications may induce the other two complications. When those three complications occur simultaneously, the probability of HE may reach 0.90 or more. The BN constructed by the tabu search algorithm can analyze not only how the correlative factors affect HE but also their interrelationships. Reasoning using BNs can describe how HE is induced on the basis of the order in which doctors acquire patient information, which is consistent with the sequential process of clinical diagnosis and treatment. Most patients with cirrhosis have different degrees of HE at varying stages of the disease, the mortality rate of which is extremely high3. The 1-year survival rate of patients who have chronic liver disease complicated with HE is 0.42, and their 3-year survival rate is 0.234. HE is divided into dominant and occult types1: dominant HE has obvious clinical manifestations, such as disturbance of consciousness, behavioral disorders, and coma5; whereas the clinical signs of occult HE are difficult to detect. Thus, prevention and early identification of HE could be a reasonable and effective way to reduce HE harm. At present, there is no “gold standard” for HE diagnosis. Clinicians can only conduct auxiliary diagnostic evaluation of HE from different angles such as liver function tests, imaging examinations, and neuropsychiatric abnormalities. Occult HE often has no clinical symptoms, and only through careful neuropsychological or neurophysiological examination can patients be found to have cognitive dysfunction. However, the neuropsychological testing process is cumbersome, can consume too much time, and is easily susceptible to the patient’s condition, educational level, and cultural background. Neurophysiological tests are inaccurate and require complex instruments, and their routine clinical applicability is relatively limited6. In short, the pathogenesis of HE is complex and diverse, and its clinical symptoms are variable and sometimes difficult to find. There are no clear diagnosis or treatment guides for it at home or abroad. Therefore, search methods for clinical diagnosis and treatment of patients with cirrhosis use patients’ clinical manifestations and factors that affect the occurrence of HE to identify HE, which has important clinical diagnostic value and is worthy of our research.Hepatic encephalopathy (HE) is a very serious complication of liver disease. It is a comprehensive symptom of central nervous system dysfunction based on metabolic disorders, caused by acute or chronic severe liver function disorder, or abnormal portal-systematic circulation8. Full and reasonable use of these data is an important basis for current big data analysis. We can use traditional logistic regression on the big data generated during clinical diagnosis and treatment of patients with cirrhosis to establish a prediction model for the incidence of HE in patients with cirrhosis9, and finally make judgments based on clinical expertise. Logistic regression requires that the independent variables be independent9, but the clinical manifestations of HE and the factors affecting the occurrence of HE are not independent, and there may be interactions that form complex networks of relationships. Therefore, it is difficult for logistic regression to accurately reveal the potential overall information in the data, and it is impossible for logistic regression to infer which factors play a role in the occurrence and development of HE. In addition, any logistic regression model that is applied must predict the probability of developing HE when the state of each variable is known. In clinical practice, the factors used for model prediction may be missing: patients may conceal certain information, or some items may not be detected in clinical diagnosis, which could lead to the failure of logistic prediction.In recent years, with the development of hospital information technology, hospital information systems contain large amounts of patient and medical data such as patient sociodemographic characteristics, clinical symptoms, physiological and biochemical indicators, and auxiliary examinations, which have formed large datasets10. BNs do not have strict requirements for statistical assumptions in the modeling process11. Although BNs do not extract variables’ main effects and interaction effects, they comprehensively reflect the complex relationships between variables according to the overall structure of the network and can accurately reveal potential overall information in the data12. BNs can use missing data to identify patients13 in a manner that reasonably reflects the sequentiality of clinical diagnosis and treatment15. The advantages of BNs in clinical applications have also been demonstrated in our previous studies. Because there were too many initial variables in this study’s initial cirrhosis data, some variables were only weakly related to the occurrence and development of HE. The direct use of a BN to establish the model leads to the selection of all variables, which is not conducive to accurate analysis. To simplify the network’s relationships and improve network performance, in this study we employed univariate and multivariate logistic regression analysis to screen for related factors of cirrhosis with HE16, and then constructed a BN to explore the related factors of cirrhosis complicated with HE, revealing the complex network relationships among related factors17. During the care process of patients with cirrhosis, we can continuously update the network probabilities according to the patient information obtained by doctors. We also adopt BNs to conduct risk reasoning about the probability of HE in patients with cirrhosis19.Bayesian networks (BNs), proposed by Judea Pear in the 1980s, reflect the potential relationships and relationship strengths between factors by constructing directed acyclic graphs (DAGs) and conditional probability distribution tablesWe collected information about patients with cirrhosis who were hospitalized in the Department of Gastroenterology, First Hospital of Shanxi Medical University from January 2006 to December 2015 and who had complete medical records. We consulted a large number of documents and carefully analyzed the cases’ structure and content and selected indicators that may be related to the progression of cirrhosis. Biochemical indicators were taken as the results of the first test within 24 hours after admission. After the data were sorted, 950 cases were effective, of which 68 were complicated by HE.There are 23 factors in the survey: demographic characteristics , lifestyle , past medical history , clinical manifestations , and biochemical indicators . All of this study’s methods were carried out in accordance with relevant guidelines and regulations.This study was approved by the First Hospital of Shanxi Medical University Ethics Committee. Informed consent was signed by all study participants or their agents. The present study was conducted according to the Declaration of Helsinki.This study’s investigators were clinicians and graduate students. Before the start of the survey, the clinicians instructed the graduate students to familiarize themselves with clinical records and to determine the location of the information to be collected in the medical records. Finally, the clinicians normalized the course of looking up medical records. Data entry was performed by double entry, and a person reviewed both sets of entries. If anomalous values were discovered during the review process, the original case was extracted and the erroneous information was corrected.20. As a reasoning method that employs probabilistic uncertainty, BNs have served in intelligent systems and have been successfully applied in medical diagnosis, expert systems, statistical decision making, learning, and prediction21.BNs are increasingly popular data-mining techniques that are used with categorical data and effective theoretical models to represent uncertain knowledge and reasoning22. The DAG’s nodes represent random variables U = {24. If there is a directional arc from X = BNs represent a set of random variables and their conditional interdependencies via a Directed Acyclic Graph (DAG)The first step of constructing a BN is to determine the nodes. To avoid including too many nodes and introducing excessive complexity to the network structure, we should initially filter the nodes in combination with prior expert knowledge or traditional statistical methods. Then, we should find the optimal model based on the tabu search algorithm. Finally, on the basis of structural learning, we should calculate the network node conditions using the maximum likelihood estimation method.25.The tabu search algorithm is a sub-heuristic algorithm that simulates human memory function. It has the characteristics of few parameters, simple structure, and robust global optimization ability. For a given current network structure, the algorithm uses three operations to generate neighborhoods without generating a network loop: add edges, reduce edges, and reverse edges, and then it searches for a local optimal solution in the neighborhood and puts it into a tabu table. The tabu table is then used to search for the local optimal solution, so that the next search is as far as possible from the current one to avoid duplication of the search process. In conjunction with the use of contempt criteria to amnesty some of the optimal solutions in the tabu table, the tabu restrictions are ignored. When these two steps iterate and search in a circular way, it results in a taboo table that is updated constantly, and ultimately, we obtain the global optimal solution1; (2) HE: refers to the “definition of HE definition, diagnosis, and quantification” standard issued by the 11th World Conference on Gastroenterology Work Group in March 2003, with the diagnosis mainly based on cirrhosis history, neurological abnormalities, liver function tests, image examination, and other auxiliary examination, excluding nerve abnormalities caused by other diseases26; (3) Hypertension: according to the World Health Organization (WHO) standards, systolic blood pressure SBP ≥ 140 mmHg and/or diastolic blood pressure DBP ≥ 90 mmHg, or having a previous history of hypertension and currently taking antihypertensive drugs27; (5) Diabetes mellitus: fasting blood glucose (FBG) ≥ 7.0 mmol/L or 2-hour postprandial plasma glucose ≥ 11.1 mmol/L or reporting diagnosed diabetes mellitus28; (6) Smoker: smoking ≥ 1 cigarette daily on average over the previous 6 months; (7) Drinker: drinking at least once per week regularly for 6 consecutive months with average intake quantity of more than 50 g29.(1) Cirrhosis: comply with the diagnostic criteria jointly revised by the Chinese Medical Association’s Infectious Diseases and Parasitic Diseases Branch and the Hepatology Branch in 2000In our study, we used Epidata to build a database and enter data. Statistical Analysis System (SAS) version 9.2 software  was used for data pretreatment. Descriptive statistics and logistic regression analysis of the data was performed with SPSS Version 22 . Significance for all statistical tests was set a priori at P < 0.05, and all P values were two-tailed. Weka 3.8.0  was used for structural learning of BNs and parameter estimation of BNs using the maximum likelihood estimation method. The BNs’ topology and conditional probability distribution tables were drawn using Netica .The total enrollment was 950 patients with liver cirrhosis, including 508 male (53.5%) and 442 female (46.5%) individuals ranging 17–88 years old, with a negatively skewed distribution. The overall median age was 58.0 years (QR = 20.3). Among the subjects, 156, 357, and 437 were aged <45, 45–59, and ≥60 years, respectively , hepatorenal syndrome (RR = 3.467), and infection (RR = 3.021) were the major risk factors for HE in patients with cirrhosis. The relationships between hepatic diabetes, poor spirits, increased total bilirubin, prothrombin time prolongation, and HE were relatively weak, with RR values ranging 2.1–2.7  curve as the evaluation index. Figure BN reasoning refers to calculating the probability of an event in a given network structure, given the available evidence. Its biggest advantage is automatic updating of network probabilities according to different degrees of information mastery.According to the patients’ visiting process and the order in which information was obtained by the doctors, we used the BN to predict which patients would have concurrent HE. When a patient with cirrhosis does not attend a clinical examination, the likelihood of HE is 0.0736 by a priori information about the BN. If a clinical examination reveals that the patient is in poor spirits and has hepatic diabetes, then the BN shows that the likelihood that the patient has HE is 0.155 . Infection, electrolyte disorders, and hepatorenal syndrome are closely related to HE: if the patient had all three conditions, the probability of concurrent HE was 0.968. The results show that the BN constructed by the tabu search algorithm has a flexible reasoning mechanism, which is helpful for disease identification and diagnosis. It has important clinical diagnostic value and could serve as an effective method to reduce harm from HE.42. BNs are also suitable for datasets with a variety of uncertain information and excessive numbers of variables11: they facilitate easy understanding and explanation through visual images of the results. However, BNs also have shortcomings: for example, the edges between variables only represent probability dependencies and do not represent causal relationships. Therefore, we cannot perform causal analysis from only the BN’s directed edges. It is necessary to combine various aspects of professional knowledge to determine causality43. The data in this study are unbalanced, which may cause incorrect classification. In future research, we intend to deal with the impact of data imbalance on the classification model at the data and algorithm levels and hope to achieve better results. For example, we can expand the scope of data collection to increase the number of HE-positive cases and continuously verify and improve the accuracy and applicability of model classification identification. In addition, this study is based on a cross-sectional survey; thus, the BN can only reveal the relevant factors related to the occurrence of HE, and the causal relationship needs further verification. Only in a BN built making use of causal relationships do the directed edges between the variables represent causal relationships44.The complex network of relationships between HE-related factors revealed by the BN is in line with clinical practice. By constructing the BN model, the clinical manifestations of patients with cirrhosis and the factors affecting their probability of occurrence of HE are used to identify the cirrhosis with HE, and the network probabilities can be updated as the doctor receives increasing amounts of patient information. Although this study lists only some of the key issues, it shows that compared with a logistic probability prediction model, the BN is more in line with the sequential process of clinical diagnosis and treatment. BNs have other advantages, including their ability to combine prior information and sample information. Subjective bias caused by the application of prior information and bias caused by using only sample information can be avoidedSupplementary informationDataset


Younger children (up to 12) are less likely to abandon the diet . In this age group non-intentional diet failure prevails, while teenagers interrupt their diet intentionally ). Currently, the most common causes of teenage diet failure are the absence of symptoms after consuming a small amount of gluten and, even more often, troublesome diet administration. Previously, the absence of peer acceptance prevailed. With this study we found that: 1. In West Pomerania, every fourth CD child does not follow GFD. 2. For years, teenagers have failed to follow GFD due to the absence of symptoms after consuming small amounts of gluten. 3. The incidence of non-intentional failure to follow GFD has significantly decreased over years, which indicates better dietary care.Celiac disease (CD) can only be treated by rigorous life-long gluten-free diet (GFD). The study included 102 mothers and their CD children treated with GFD for at least two years. Frequency and cause of diet failure in children treated at present (54 children) and 10 years ago (48 children) were compared. Dietary adherence was evaluated serologically (tTG), while diet management difficulties were examined by means of a questionnaire. The study shows that one-third of patients fail to follow GFD, more often 10 years ago than now (40% vs. 26%;  Celiac disease (CD) is a genetically conditioned, immunologically mediated chronic intestinal disease, in which in genetically predisposed people the consumption of gluten leads to the disappearance of intestinal flora . This reThe most reliable method to control GFD adherence are serological tests and small intestine biopsy . It is rSome researchers suggest using nutritional history to assess the GFD adherence. However, it does not allow to identify patients who non-intentionally fail to adhere to GFD, therefore its importance for monitoring procedures is low. Leffler et al. proposed a seven-point questionnaire to identify patients who did not adhere to GFD . The test assesses their knowledge of the disease, which, according to Leffler, is highly correlated with serological test results . In 2017Adherence to GFD is troublesome . The patIn the opinion of some authors, the following factors contribute to the better adherence to GFD: good knowledge of the disease and its treatment, higher education level, better social situation of the family, female sex, young age, high self-esteem, good grades at school, good availability and labelling of products, good contact with a doctor and a dietitian, and finally, membership in the Coeliac Society ,11,12,13The factors responsible for not adhering to GFD are: poor taste of gluten-free products, their high price and low availability , the patient's adolescence, the absence of immediate symptoms after consuming small amounts of gluten, and low awareness of the disease ,11,13. Better awareness of the factors that have significant impact on the GFD adherence can improve the supervision of CD patients.The aim of this study was to determine the incidence and causes of non-adherence to GFD by children with CD treated now and 10 years ago.The first analysis of the causes of failure to adhere to GFD was carried out in 2006–2007 with a view to improving the effectiveness of CD treatment, as it had been observed that many patients discontinued GDF after years of adherence. In recent years, GFD has become popular in Poland and therefore the conclusions concerning the patients’ failure to adhere to GFD 10 years ago are no longer relevant. Unfortunately, it has been observed that teenagers still tend to discontinue their GFD. Therefore, in the 2016–2017 study, the questions about the reason for failure to follow GFD were again included in the CD patients’ medical history.The study covered 102 children (64 girls) with coeliac disease (CD) treated with GFD. All patients were diagnosed in accordance with the applicable criteria: elevated antibodies anti–endomysium (EMA) and anti-tissue transglutaminase (tTG) as well as duodenal atrophy of intestinal mucosa . The patients were recruited during their GFD treatment, two years or more after the diagnosis, during control tests performed routinely twice a year. The mean time of the GFD treatment before their inclusion in the study was 104 months (28 months–208 months). During the medical interview, patients made a declaration concerning their dietary adherence. If they reported that they did not follow the diet, they were asked to give reasons. Five probable causes were specified: poor taste of the diet, its high price, troublesome dietary regime , difficulties in relationships with peers (due to being rejected by the peer group) and the absence of symptoms after consuming gluten products. Each patient was asked to choose which of the reasons was the most relevant to them, i.e., which led to deliberately abandoning of GFD. In the case of the youngest children (under seven years of age), dietary declarations were made by their guardians . Older children with CD and their guardians jointly chose the most important reason for their failure to follow GFD.The tests performed during each control visit included serum assays of tissue transglutaminase (tTG) in the IgA class. The presence of tTG antibodies in serum confirmed that the CD patient had consumed gluten. Patients who claimed to adhere to GFD and in whose blood tTG antibodies were found were considered as those who non-intentionally failed to adhere to GFD. In 33 children who did not follow GFD, the average IgA-tTG concentration was 126 RU/mL (264 RU/mL–22 RU/mL). In children adhering to GFD the IgA-tTG concentration was below 20 RU/mL. Patients were divided into two groups: children (0–12 years old) and teenagers (13–18 years old) . The fret-test was used for independent trials.EMA was determined by indirect immunofluorescence test (standard < 1:10), while IgA-tTG was assayed by ELISA (standard < 20 RU/mL). The obtained results were subjected to statistical analysis. The Student’s The survey did not require the permission of the PUM  Bioethical Committee. The relevant opinion was obtained from the Chairman of the Committee in 2006. All subjects had given their informed consent for inclusion before they were included in the study.p < 0.05) (p < 0.05). Younger children (up to 12 years) were less likely to miss the diet . At that age, the incidence of non-intentional GFD failure was larger: 4% at present (1 of 24 children) and 19% previously (5 of 26 children)  . Those whildren) . Teenagehildren) . Currenthildren) . In 2006hildren) .The analysis shows that currently every fourth child with CD does not adhere to GFD . Ten yeaCurrently, teenagers are five times more likely not to adhere to GFD than younger children (40% vs. 8%). In 2006–2007, teenagers consumed gluten only twice as often as younger children (54% vs. 27%). Similar observations were made by other researchers ,18. WhatBoth 10 years ago and now, the majority of CD teenagers intentionally fail to adhere to GFD , which hIn younger children (<3 years), the consumption of even the smallest amounts of gluten usually result in intestinal symptoms, most commonly abdominal pain or abnormal stools. Teenagers, seeing that such a co-incidence no longer occurs, start to believe that they can consume gluten in small amounts. There are patients who are able to precisely define the amount of gluten that cause the symptoms. Therefore, they try to consume smaller amounts, which in their opinion does not harm their health. In this group of patients, dietary errors are caused by insufficient knowledge of CD and its treatment. They need to be informed about the CD again. It seems that such information should be provided to every juvenile patient once they enter adolescence. It should be particularly pointed out that, at this age, eating small amounts of gluten may not cause such intestinal discomfort as previously, but it still remains detrimental to their health. We try to persuade these patients to adhere strictly to GFD by showing them that their growth rate has been slowed down, anemia induced by iron deficiency has occurred, bone density has decreased , and antibodies indicating CD activity have appeared in serum. In some cases, we repeat endoscopic tests of fragments from the duodenum in order to show that intestinal villi atrophy has reoccurred in their duodenum. We explain that these are the complications due to the young patient's failure to adhere to GFD and that they have appeared independently from the absence of intestinal discomfort. Systematic monitoring of teenagers' dietary adherence is absolutely essential, even if they have had a history of strict GFD adherence. Other authors also believe that the lack of immediate symptoms after consumption of small amounts of gluten is an important factor in teenagers' failure to adhere to GFD .Ten years earlier, in 2006–2007, the main cause of non-adherence to GFD by teenagers was peer rejection due to their different dietary regime. These children were stigmatized by other children and isolated from the group. In order not to be socially excluded, teenagers with CD intentionally consumed gluten products while in social situations. None of these children changed their behavior throughout the treatment period (up to 18 years), despite having been informed about the adverse effects of such behavior. Similar teenagers’ attitudes have also been described by other authors ,6,16,19.In our 2016–2017 study, none of the children reported the above as a reason for non-adherence to GFD. This is a consequence of a change in social customs. Today, GFD has become popular in Poland and it is used by many people who do not suffer from CD. Similar trends have also been observed in other countries where the frequency of GFD consumption far exceeds the number of CD patients ,21,22.Younger children have never reported this problem. In kindergartens and schools, they consumed gluten-free products and that fact did not have any negative impact on their peer relations.At present, just like 10 years ago, the second most frequent reason for non-adherence to GFD is its troublesome administration, as reported by both younger children and adolescents . By the When buying gluten-free products, patients can benefit from a mobile application containing an inventory of gluten-free products available in a given country . Such anIn our studies none of the CD patients gave a high GFD price as the main reason for not following it. That was another, but not the most important, reason. During the 2006–2007 study, all Polish children with CD received a cash allowance to buy GFD. Unfortunately, today the allowance is not available to the majority of children. Many authors point out that the high price of GFD is an important reason for not adhering to GFD by many patients ,13,26. INone of the examined groups of children indicated the illegible labelling of the gluten-free products as the most important reason for not adhering to GFD, which has also been reported by other researchers ,8,13. Strict adherence to GFD is most common among patients with greater knowledge of their disease . They arOver the last 10 years, an increase in the number of children with celiac disease adhering to the gluten-free diet has been observed in the West Pomeranian region. Similarly, to other countries, today every fourth child with celiac disease does not follow a gluten-free diet. For years, it was teenagers who most often failed to adhere to the gluten-free diet. The lack of awareness of the course of the disease, the increasing independence from parents, the need to buy food and prepare meals for themselves, the need to better organize everyday life as well as the environmental pressure are the factors resulting in more frequent failure to follow GFD. Teenagers require psychological support and repeated instruction explaining to them that they cannot eat even the smallest amounts of gluten, even though it does not cause any intestinal symptoms at this age, but unfortunately is harmful to their health.Over the years, the frequency of non-intentional non-adherence to the gluten-free diet has significantly decreased as a result of better dietary care.Systematic medical and dietary supervision contributes to more effective treatment of children with celiac disease, which in turn ensures their proper development and prevents complications.


In the past, only people diagnosed with celiac disease, approximately 1% of the population, avoided gluten consumption through all their meals. However, popular media often now mistakenly present gluten-free foods as being a healthier choice, and more people have now concluded that gluten is a harmful part of the diet. A review of literature on gluten-free diets, gluten sensitivity, celiac disease, and attitudes toward gluten consumption was undertaken to examine the prevalence and consequences of adopting a gluten-free diet and to provide guidance to healthcare practitioners whose patients are now often adopting this diet without medical input. Aside from celiac disease, nonceliac gluten sensitivity (NCGS) occurs in those persons in which gluten ingestion leads to symptomatic manifestations in the absence of celiac disease or wheat allergy but who report a remission of certain symptoms after removing gluten from their diet. However, it was been shown that a large percentage of people who claim NCGS do not feel those manifestations under a double-blind challenge to gluten. Moreover, some parents, believing that ingesting gluten is detrimental for their health, adopt gluten-free diets for their children. A review of existing data shows that there are detrimental effects to going gluten free, including loss of the dietary fiber, deficiencies in dietary minerals and vitamins, and potential heavy metal exposure. Healthcare practitioners should query patients about their dietary choices, and in cases of questionable adoption of gluten-free diet, patients and parents are educated about the detriments of a gluten-free diet, and in cases where patients continue to insist on gluten-free foods, referrals to nutritional counseling are warranted in order to minimize potential harm. There is an increasing body of evidence that a significant proportion of individuals, including not only persons with celiac disease (CD) or nonceliac gluten sensitivity (NCGS), are following a gluten-free diet (GFD). “Gluten free” was defined by the US Food and Drug Administration in 2013 as foods containing less than 20 parts per million of gluten or 20 mg/kg , compareGluten is a high-molecular-weight seed storage protein commonly found in grass-related grains, such as wheat, barley, and rye. The natural role of these seed storage proteins is to nourish seeds during flowering and germination, thereby contributing to the successful reproduction of the species . Gluten Celiac disease (CD) affects approximately 1% of the population –10 and iNonceliac gluten sensitivity (NCGS) occurs in those persons in which gluten ingestion leads to symptomatic manifestations in the absence of celiac disease or wheat allergy (WA) but who report a remission of certain symptoms after removing gluten from their diet , 17. SymThe Salerno Expert's Criteria provide guidance on how to test someone for NCGS. It is based on symptoms but follows a single- or double-blind placebo-controlled challenge . A key eLioneti et al.  found thbonafide need to avoid gluten. It has even been suggested that it may be more appropriate to describe these patients as having “nonceliac wheat sensitivity” or “self-reported NCGS” in the absence of clinically confirmed gluten insensitivity [Diagnosis of NCGS is very difficult, mostly because of the lack of validated diagnostic criteria and the high nocebo and placebo effects of gluten . The trusitivity , 26.For those individuals that truly suffer from NCGS, the manifestations can be intestinal or extraintestinal. Intestinal manifestations include abdominal pain, bloating, and stool inconsistency, while extraintestinal manifestations can include fatigue, joint and muscle pain, and limb numbness. Moreover, neuronal manifestations can also present due to ingestion of gluten in some people. These manifestations include foggy mind, headache, gluten neuropathy, gluten encephalopathy, and gluten ataxia .Numerous studies have demonstrated that adherence to a GFD significantly increases the cost of food for adherents, with prices 2 to 3 times greater than for similar nongluten-free products –30. OverGFD can lead to nutritional deficiencies of macronutrients and micronutrients. Gluten-free foods, when compared to equivalent wheat-based foods, show deficiencies in minerals, including calcium, iron, magnesium, and zinc, and in vitamins, including vitamin B12, folate, and vitamin D, as well as significantly reduced fiber . A 2013 Notably, some food components known to be detrimental to health are higher in the GFD than in a regular diet. For example, a GFD increases dietary exposure to arsenic , and theFor patients diagnosed with CD, a gluten-free diet is the only treatment. Ingestion of gluten in these genetically predisposed individuals results in a T-cell-mediated immune reaction, leading to villous atrophy and clinical symptoms. Avoiding gluten prevents this response, and as such, for CD patients, a gluten-free diet is critical to their well-being.For sufferers of CD, the increase in popularity of a GFD among the general population has led to dramatically more selections of foods devoid of gluten, which are likely more palatable and more likely to be enriched with vitamins and minerals to meet the demands of an expanded marketplace. Also, more studies are aimed at finding those food sources that are still gluten free but higher in nutrients missing in a GFD.For individuals who do not suffer from CD, benefits of a GFD are less obvious. The positive outcomes of a nocebo/placebo effect for those with NCGS, even if it is not the gluten component of wheat that leads to their symptoms, cannot be discounted. For some individuals, this may justify the detriments of a GFD.The benefits of GFD continue to have an increased interest among researchers and practitioners working on the improvement of behavior in children with autism and other spectrum disorders. Even though some research informed that there was not significant evidence of improvement of functioning in children with autism, GFD was reported as well tolerated and safe . Lee et As outlined above, there is a risk of nutritional deficiencies when either adults or children are subject to a GFD. As such, it is imperative to have the right diagnosis before placing any child on a GFD. For children with celiac disease, there is no other diet to follow than the GFD, yet diagnosis of CD requires that an individual continue to eat gluten prior to the diagnosis. After diagnosis, having a diet without gluten will help those children to heal their intestine and absorb more nutrients than if they are ingesting gluten.In children that are diagnosed with NCGS, extra precaution needs to be taken because of the dangers of nutritional deficiency and the difficulty in achieving an accurate diagnosis absent a double-blind challenge, which is not typically achievable through a primary care healthcare provider. Tanpowpong et al.  found thThus, the actual prevalence of NCGS does not appear to justify the commonality of a GFD in children, given the potential detriments of a GFD. In fact, there is currently no evidence to support the imposition of a GFD in asymptomatic children in the absence of CD or documented wheat allergy .It is necessary for pediatricians, nurse practitioners, and other health professionals working with children to query parents about their choice of diet for their children. As a sizable slice of the population has misidentified limiting gluten intake as being beneficial to their health, it seems to follow that those who subscribe to this belief and who become parents may feel similarly about what their children eat. It is common during routine exams for healthcare practitioners to ask about whether kids eat vegetables and fruits to get a sense of their diet. It is also important to establish whether kids eat grains to determine whether parents might be unnecessarily limiting their children to a GFD.Although the long-term health effects of a GFD have not been established, a systematic review of prior studies has identified increased total cholesterol, high density lipoprotein, fasting glycemia, and body mass index as consistent findings . SimilarFor parents of children with CD, it is a common practice for gastroenterologists and/or nutritionists to consult with parents to teach them the limitations of a GFD and to ensure that their children not only avoid gluten but also to teach parents how to secure the nutrients that are likely to be missing from their child's diet. Similarly, with the risk of a GFD in the general population, healthcare practitioners need to proactively question whether their patients, whether children or adults, are following a gluten-free diet of their own volition.If patients, either adults or children, are suffering from symptoms that may be associated with CD, then referral to a gastroenterologist for proper diagnosis is critical. However, in the absence of symptoms, or vague descriptions of “gluten allergy” or other unrecognized disorders, patients who follow a GFD first need education on the true prevalence of CD and NCGS. They need to be taught of the need to secure sources of nutrients that may be limited in a GFD, and they need to know that following a GFD blindly may be harming themselves or their children. In the end, only persons with CD, WA, or confirmed NCGS should follow a GFD, and they should do so under medical supervision.Adults who self-administer a GFD and cite physical symptoms should be referred for CD testing. Adults who do not have CD but continue to insist on a GFD should be referred for nutritional counseling to ensure they are meeting their dietary needs and avoiding unnecessary hazards. Multivitamin/mineral and fiber supplements are a good idea for anyone attempting to avoid gluten to ensure adequate nutrition and to ensure proper bowel function.For children who are put on a GFD, often by well-meaning parents, in the absence of a clinically relevant diagnosis, it is important that the parent be educated on the risks of a GFD, the need for CD testing if the parents claim symptoms in the child, and the potential hazards to their child of a GFD. If they insist, a referral to nutritional counseling is warranted.While celiac disease patients must permanently remove gluten from their diet, the literature suggests that many individuals who claim to suffer from NCGS may in fact have no actual sensitivity to gluten although it is possible they are sensitive to other components of wheat. Alternatively, these individuals may be benefitting from a nocebo/placebo effect when it comes to adopting a GFD. It is likely, however, that the majority of persons adopting a GFD have no medical basis for doing so.The choice to adopt a GFD is often made by individuals without medical input, in the belief that they are adopting a healthier diet. Unfortunately, choosing GFD, irrespective of whether it is medically indicated, generally entails increased food costs, a decrease in fiber consumption, potential decreases in mineral and vitamin consumption, including calcium, magnesium, zinc, vitamin B12, folate, and vitamin D, and potentially increased exposure to dietary hydrogenated and saturated fatty acids, and arsenic. Clearly, adopting a GFD does not come without risk.In the end, only persons with CD, WA, or confirmed NCGS should follow a GFD, and they should do so under medical supervision. For individuals who insist on a GFD in the absence of a diagnosis that suggests its benefits outweighs its risks, nutritional counseling is recommended to ensure that these individuals can minimize the risks to themselves while still choosing the diet they prefer.


There is accumulating evidence that deregulated Notch signaling affects cancer development, and specifically pancreatic cancer (PC) progression. Notch canonical and non-canonical signaling has diverse impact on PC. Moreover, the actions of RBP-Jk (nuclear partner of activated Notch) independent of Notch signaling pathway seem to affect differently cancer progression. Recent data show that in PC and other cancer types the adipokine leptin can modulate Notch/RBP-Jk signaling, thereby, linking the pandemic obesity with cancer and chemoresistance. The potential pivotal role of leptin on PC, and its connection with Notch signaling and chemoresistance are still not completely understood. In this review, we will describe the most important aspects of Notch-RBP-Jk signaling in PC. Further, we will discuss on studies related to RBP-Jk-independent Notch and Notch-independent RPB-Jk signaling. We will also discuss on the novel crosstalk between leptin and Notch in PC and its implications in chemoresistance. The effects of leptin-Notch/RBP-Jk signaling on cancer cell proliferation, apoptosis, and drug resistance require more investigation. Data from these investigations could help to open unexplored ways to improve PC treatment success that has shown little progress for many years. PC is one of the most malignant and chemotherapy-resistant tumors. That is mainly due to the lack of effective diagnosis at an early stage of tumor development and ineffective therapy. Resectable tumors at detection time comprise a low percentage of PC. Majority of PC are treated with non-specific target therapies, i.e., chemotherapeutic drugs, but its effectiveness is limited due to the dense tumor stroma (desmoplasia) and the acquisition of drug resistance. Multiple mechanisms are involved in PC chemoresistance, including aberrant gene expression, deregulation of key signaling pathways, development of the epithelial-mesenchymal transition (EMT), cancer stem cells and desmoplasia .A factor involved in cancer chemoresistance is the activation and deregulation of the Notch signaling pathway. Notch signaling has been recently linked to 5-FU resistance in PC . This emNgn3 gene, which antagonizes Hes1 and suspends Notch control , EMT  and PC stem cells (PCSC) .The expression of ABC family of proteins impairs the effects of chemotherapeutics by increasing the efflux of drugs from the cells. Several ABC transporters were upregulated in PC compared to non-neoplastic tissue . AdditioEMT induces higher migratory, invasive, anti-apoptotic and extracellular matrix degradation capacities that characterized the mesenchymal phenotype. EMT was associated with overexpression of Notch signaling components that correlated to higher migration and invasion of PC cells. Notably, EMT phenotype was reverted via Notch inhibition, which reduced the expression of ZEB1, Snail, Slug and Vimentin in PC cell lines ,71. MoreDCAMKL-1 is a microtubule-associated kinase that downregulates pluripotent cancer stem cell markers , but induces microRNA200 (miRNA200a). DCAMKL-1 was overexpressed in a subset of PanIN and PC cells. EMT was inhibited via knockdown of DCAMKL-1 that also inhibited Notch1 via miR-144 .The Notch signaling pathway is a component of the stem cell signaling network (SCSN) together with Wnt, FGF, BMP and Hedgehog signaling pathways . SCSN isCancer stem cells are commonly more resistant to chemotherapeutics, which preferably target the bulk of the tumor. Therefore, chemotherapeutic actions spare stem cells, which show higher expression of ABC proteins, detoxification proteins and survival pathways (NF-κB and PI-3K) and diminished apoptosis rate . EarlierA wide-used PC drug, gemcitabine, increased PCSC expressing CD24+ and CD133+, stemness-associated genes , cell migration, chemoresistance, and tumorigenesis . GemcitaMicroRNAs (miRNA or miR) are noncoding endogenous RNA of 14-24 nucleotides that can regulate protein expression at the post-transcriptional level. Many studies have found strong correlations between deregulated miRNA and cancer. Decreased expression of microRNA (miRNA34 and miR200) families was early associated with PC progression and PCSC . It has MiRNA200 family , miR429, and miR141 have been implicated in PC chemoresistance. miR200c expression correlated with chemoresistance and less cancer invasion via repression of cancer stem cell self-renewal and differentiation, inhibition of EMT and attenuation of apoptosis. In PC, miR200 repressed self-renewal and differentiation of stem cells, and inhibited EMT by interacting with ZEB1/2 and the Notch pathway . MoreoveAdipokines (leptin and adiponectin) are adipose tissue-derived cytokines that can act in opposing manner: leptin contributes to a pro-tumorigenic phenotype, while adiponectin acts as anti-tumorigenic factor. The pluripotential actions of leptin as an inflammatory, mitogenic and proangiogenic factor, have been linked to cancer cell proliferation, recurrence, tumor angiogenesis and chemoresistance. We firstly reported that leptin induces the expression of Notch family components in breast cancer, which was linked to IL-1 signaling ,92,93. LRNA knockdown and pharmacological inhibitors of leptin signaling significantly abrogated the activity of reporter gene-luciferase CSL (RBP-Jk) promoter, showing that its activity was linked to JAK2/STAT3, MAPK, PI-3K/mTOR, p38 and JNK signaling pathways. DAPT (a GSI) and siRNA RBP-Jk abrogated leptin’s effects on cell proliferation/migration, Notch, IL-1 and VEGF/VEGFR-2 . FurtherIt is known that leptin and Notch signaling mediate the activation of cancer stem cells that can affect drug resistance ,54,95. WOur published data also show that leptin is a survival factor for PC cells treated with chemotherapeutics. Interestingly, 5-FU’s toxic effects on PC cells were impaired by leptin. PC cells treated with 5-FU in presence of leptin showed significantly higher proliferation rate and colony forming ability. In addition, these cells expressed higher levels of EMT, pluripotency and PCSC markers. Notably, leptin increased the expression of ABC proteins (ABCC5 and ABCC11), and Notch in PC cells treated with 5-FU. Moreover, leptin impaired 5-FU-induced apoptotic effects by increasing RIP and Bcl-XL and reducing Caspase-3 activation, PARP degradation and Bax expression. Remarkably, these leptin effects on PC cells treated with 5-FU were dependent of Notch signaling . In addiThe Notch signaling pathway can be inhibited pharmacologically, via GSI, anti-Notch antibodies, etc. GSI have been tested in clinical trials for many cancers, including PC. The agent RO4929097 in combination with gemcitabine was safely tolerated, achieved clinical antitumor activity and more than four months stable disease in PC, tracheal, and breast cancers . Dual trPC has also been treated with monoclonal antibody-based therapies. A fully humanized anti-Notch2 and Notch3 antibody (Tarextumab or OMP-59R5) reduced the growth of PC xenografts in mice when combined with cytotoxic drugs. Tarextumab  in combination with gemcitabine and nab-paclitaxel (Abraxane) was applied to untreated metastatic PC patients. The monoclonal antibody-based therapy allowed 5.6 months of median time of progression-free survival and 11.6 months of overall survival. PC patients with high levels of Notch3 showed better response . In anotRecent reports suggested that inhibitors of other components of Notch signaling , could open new targeted therapy opportunities. Indeed, the inhibition of ADAM10 (overexpressed in PC) via a calcium channel blocker (Fendiline) significantly reduced proliferation and tumorigenesis of PC cells . AdditioObesity, characterized by high levels of leptin, is a PC risk factor . In viewPre-clinical data from our laboratory have shown a potential for leptin signaling inhibition  as adjuvant for chemotherapeutic treatment of PC ,52,96,97PC dismal survival and poor treatment outcomes have been steady over years. Reported data underline the role of Notch in PC and other cancers development. However, the activation and regulation of Notch signaling in PC are still not completely understood. The specific contribution and regulation of Notch canonical and non-canonical signaling as well as the less investigated RBP-Jk signaling independent of Notch to oncogenesis and progression of PC remain scientific challenges. Leptin is a factor that could affect Notch signaling. Leptin’s levels are increased in obesity that is pandemic and strongly linked to incidence of PC and other cancers. Analysis of recent data shows strong relationships between leptin and Notch/RBP-Jk signaling that could open new opportunities of research and potentially increase the success of PC treatment and decrease chemoresistance. The crosstalk between leptin, Notch and RBP-Jk signaling in PC warrants further investigations.


Biological soil crusts (biocrusts) accommodate diverse communities of phototrophic and heterotrophic microorganisms. Heterotrophic protists have critical roles in the microbial food webs of soils, with Cercozoa and Endomyxa often being dominant groups. Still, the diversity, community composition, and functions of Cercozoa and Endomyxa in biocrusts have been little explored. In this study, using a high-throughput sequencing method with taxon-specific barcoded primers, we studied cercozoan and endomyxan communities in biocrusts from two unique habitats (subarctic grassland and temperate dunes). The communities differed strongly, with the grassland and dunes being dominated by Sarcomonadea (69%) and Thecofilosea (43%), respectively. Endomyxa and Phytomyxea were the minor components in dunes. Sandonidae, Allapsidae, and Rhogostomidae were the most abundant taxa in both habitats. In terms of functionality, up to 69% of the grassland community was constituted by bacterivorous Cercozoa. In contrast, cercozoan and endomyxan communities in dunes consisted of 31% bacterivores, 25% omnivores, and 20% eukaryvores. Facultative and obligate eukaryvores mostly belonged to the families Rhogostomidae, Fiscullidae, Euglyphidae, Leptophryidae, and Cercomonadidae, most of which are known to feed mainly on algae. Biocrust edaphic parameters such as pH, total organic carbon, nitrogen, and phosphorus did not have any significant influence on shaping cercozoan communities within each habitat, which confirms previous results from dunes. Globally distributed biocrusts entail an aggregation of soil particles and host diverse communities of terrestrial phototrophic and heterotrophic organisms ,2. HeterBiocrust consumers such as heterotrophic protists, rotifers, tardigrades, nematodes, and micro-arthropods are vital players in the soil food web . While pCercozoa and Endomyxa are considered as dominant groups of protists in soil ecosystems ,11,12,13Several studies were done on the diversity and community composition of Cercozoa in various soil habitats ,16,17,18The current study continues the previous work of Khanipour Roshan et al.  by focusIn this study using a high-throughput sequencing approach with specific barcoded primers , we asseThe studied areas are located in Germany  and Iceland. Biocrust samples were collected in dunes along the Baltic Sea shoreline of the German Federal State of Mecklenburg-Western Pomerania. The Baltic Sea coastline is influenced by a temperate continental climate. Climate parameters for the area were obtained from two nearby meteorological stations (Warnemünde and Karlshagen) . Total pThe sampling locations in Iceland were established around Litla-Skard, which is situated in the west of the island, about 100 km north of Reykjavík. Iceland is a volcanic island with a subpolar oceanic climate featuring an annual average temperature of 3.1 °C. The average annual precipitation in the form of rain and snow is 930 and 100 mm, respectively (data are from the climate station Borgarnes) . The vegBiocrusts sampling took place in Germany  and Iceland . Along the Baltic Sea shoreline of the German Federal State of Mecklenburg-Western Pomerania, twenty samples of cyanobacterial-algal biocrusts were collected in coastal dunes on sun exposed slopes  were sampled  29]. Th. Th29]. For biocrust sampling, a column soil sampler (collector) was pushed 1 cm deep into the respective biocrusts and carefully lifted. Samples were gently transferred from the sampler to centrifuge tubes and immediately frozen in the field.DNA was extracted from about 0.25 g biocrust by using the MoBio Power Soil DNA isolation kit  and kept frozen until processing. To amplify a DNA fragment of about 350 base pairs from the variable nuclear 18S V4 region, a two-step PCR with barcoded primers specifically designed for Cercozoa was performed . In the As an internal standard, a mock community from known species of Cercozoa (ten cultures) was created for the bioinformatics pipeline . Aft−50 and keeping only the best hit, sequences were identified in the PR2 database [Paired reads were assembled using MOTHUR v.3.9 , allowindatabase , and nondatabase  as impledatabase ). Sequendatabase , with abdatabase . Finallydatabase . In thei2 (0.01 M) solution. Total organic carbon (TOC), total nitrogen (TN), and total phosphorus (TP) were determined from dried and milled biocrust material. TOC and TN were determined after acidification (10% HCl) to remove inorganic C, using a CHNS-Analyzer  [2S2O8 (0.2 mM) and 5 mL 9 N H2SO4 (50%) in 100 mL ultrapure water) in an oven (90 °C) for 24 h. After neutralization with 1 N HCl, the samples were alkalized with nitrophenol (0.8 g in 100 mL distilled water) (two to three drops), titrated with NaOH (1 M), and HCl (1 M), filled up to 100 mL with ultrapure water and filtered . With a spectral photometer , TP was measured at 885 nm [In the current study, the following chemical properties were determined in the biocrust samples. The pH was measured in a 1:2.5 soil/aqueous CaClGermany) ,37. TP wGermany)  after dit 885 nm  in compat 885 nm  . At a high taxonomic level, 85% of the sequences could be assigned to Cercozoa, 13% to Endomyxa, and 2% to novel-clade-10-12. Both habitats, the subarctic grasslands and temperate coastal dunes, shared 96.3% of OTUs, with only two unique OTUs (1.8%) detected in each , but thestomidae .At the family level, in the dune biocrusts, Rhogostomidae and Allapsidae occurred in almost similar proportions of 14% and 15%, respectively, followed by Euglyphidae (10%), the CCW10-lineage, unknown Thecofilosea, and undescribed Glissomonadida (8% each) . In contp = 0.03) (p = 0.02)  (p > 0.05) , H’dunes = 2.14, H’grassland = 2.43)  a. While  = 0.02) b. Variatctively) . At the  > 0.05) .2 = 0.14, p = 0.001, 2 = 0.08, p = 0.02) and the CN ratio  have a significant influence on the cercozoan and endomyxan community composition between these two habitats. Noteworthy, the factors organic C and CN ratio also differed clearly between Iceland and the German dunes . Biocrusts of the grasslands were dominated by bacterivores (70%) , while only 31% of the cercozoans in dune biocrust were bacterivores. The communities showed a comparatively high level of omnivory  and eukaryvory . Omnivory was mostly related to the families Rhogostomidae, Euglyphidae, and Cercomonadidae, and eukaryvory to the families Fiscullidae, Protaspa-lineage and, Leptophryidae. Omnivorous and eukaryvorous Cercozoa included potential and known algivores. Plant parasites (Phytomyxea) constituted 4% of the grassland communities, while in the dune communities, this trophic group was found in very low abundance . According to Seppey et al. [Rhogostoma may be the most abundant terrestrial algivorous protists. Therefore, it is not surprising that the third-most abundant cercozoan taxon in the present study was the algivorous Rhogostomidae (Thecofilosea), which was found in dune biocrusts with a proportion of 14%.In comparison to other habitats ,18,19,30y et al. , speciesLess abundant thecofilosean taxa in our survey were undescribed species of the Protaspididae, the CCW10_lineage, Fiscullidae, and Pseudodifflugiidae. Although the prey range of these taxa has not yet been determined, from their evolutionary position, it is likely that they feed on algae as well.In comparison with other studies ,67, the Another possible explanation of competition among taxa is the ecological preferences and adaptations of Cercozoa to habitat conditions, in a way that the theca (shell) of Thecofiloseans well protects against evaporation. This has long been hypothesized and was finally supported by a strong negative correlation between soil moisture and the abundance of sequence reads assigned to shell-bearing amoebae in Fiore-Donno et al. . As a coIn the current study, another dominant group belonged to the omnivorous order of Euglyphida . TraditiSpongospora nasturtii, Spongospora sp., and Plasmodiophora brassicae could be identified [The exceptionally low abundance of plant parasites sequences (Phytomyxea), especially in coastal dunes, can be explained by the low abundance of roots from vascular plants, whereas in the grassland with some coverage of higher plants, low abundances of OTUs assigned to entified .In this study, we discovered diverse communities of Cercozoa and Endomyxa in biocrusts from two contrasting habitats using a high-throughput sequencing method. In both habitats, we observed similar OTU richness but different alpha and beta diversity, and evenness. The composition of cercozoan and endomyxan communities within each habitat did not change under the influence of biocrust chemical properties, as at higher trophic levels, dependency on edaphic factors decreases. This suggests that other factors are responsible for shaping the community, such as prey communities or other habitat properties . As the OTU richness was found to be similar, it is likely that different microclimatic conditions in dunes and grassland caused most of the detected taxa to grow in different proportions. At a geographic level, soil organic carbon and CN ratio influenced the cercozoan and endomyxan community composition between the grassland and dune habitats, which suggests these factors as possible drivers for the observed difference.The dominance of bacterivores in grassland biocrusts suggests bacteria as the main food source, which growth is known to be stimulated by root exudates of the dominant vegetation. It seems that in dunes, a variety of food sources such as algae created an opportunity for a more pronounced community of algivores. For a better understanding of eukaryvory in biocrust food webs, the co-occurrence of algal and other eukaryotes  in different crust types with protists deserves more attention in future studies.


Ser129α-Syn) is known to be closely associated with α-Synucleinopathies, especially Parkinson's disease (PD). The present study aimed to explore the α-Syn accumulation and its phosphorylation in the enteric nervous system (ENS) in patients without neurodegeneration. Patients who underwent colorectal surgery for either malignant or benign tumors that were not suitable for endoscopic resection (n = 19) were recruited to obtain normal intestinal specimens, which were used to assess α-Syn immunoreactivity patterns using α-Syn and pSer129α-Syn antibodies. Furthermore, the sub-location of α-Syn in neurons was identified by α-Syn/neurofilament double staining. Semi-quantitative counting was used to evaluate the expression of α-Syn and pSer129α-Syn in the ENS. Positive staining of α-Syn was detected in all intestinal layers in patients with non-neurodegenerative diseases. There was no significant correlation between the distribution of α-Syn and age (p = 0.554) or tumor stage (p = 0.751). Positive staining for pSer129α-Syn was only observed in the submucosa and myenteric plexus layers. The accumulation of pSer129α-Syn increased with age. In addition, we found that the degenerative changes of the ENS were related to the degree of tumor malignancy (p = 0.022). The deposits of α-Syn were present in the ENS of patients with non-neurodegenerative disorders; particularly the age-dependent expression of pSer129α-Syn in the submucosa and myenteric plexus. The current findings of α-Syn immunostaining in the ENS under near non-pathological conditions weaken the basis of using α-Syn pathology as a suitable hallmark to diagnose α-Synucleinopathies including PD. However, our data provided unique perspectives to study gastrointestinal dysfunction in non-neurodegenerative disorders. These findings provide new evidence to elucidate the neuropathological characteristics and α-Syn pathology pattern of the ENS in non-neurodegenerative conditions.Alpha-synuclein (α-Syn) is widely distributed and involved in the regulation of the nervous system. The phosphorylation of α-Syn at serine 129 (p Alpha-synuclein (α-Syn) is widely distributed in the nervous system and is involved in the regulation of synaptic plasticity, as well as the packaging and trafficking of vesicles  has been shown to be the most abundant form in intracellular inclusion bodies in the brain of patients with PD , leading to neurotoxicity . All the involved patients  attended Sun Yat-sen Memorial Hospital. Written informed consent for this research was obtained from all individuals prior to surgery. Intestinal specimens were obtained from patients who underwent colorectal surgery for malignant or benign tumors that were not suitable for endoscopic resection. A 2 cm-wide sample of the intestinal tract was taken as the initial specimen within a safe distance (10 cm) from the tumor , α-Syn and pathologic α-Syn were employed to assess their distribution patterns. For immunohistochemical staining, the initial tissue was immersed in 4% neutral formalin solution for fixation within 1 h after tissue extraction. All specimens were fixed for more than 48 h. Then, a 1.5 cm × 1.5 cm full-thickness intestinal sample was selected for paraffin embedding and preparation. Continuous sectioning was performed, and the thickness of each section was 4 μm.After baking , xylene dewaxing, and gradient alcohol (100–90–75%) dehydration, antigen retrieval of the samples on the slides was performed in a microwave oven with high fire for 10 min and medium-high fire for 10 min, using sodium citrate solution . Then, the sections were blocked using an endogenous peroxidase blocker (10 min) and 5% goat serum (15 min). They were then incubated with primary antibodies  antibody, 1:500, MABN826, Millipore, Billerica, MA, USA) at 4°C overnight, followed by incubation with a biotin-conjugated secondary antibody  for 15 min at room temperature (RT) and horseradish peroxidase (HRP)-labeled streptavidin  for 10 min at RT. After reaction with 3,3′-Diaminobenzidine (DAB) peroxidase substrate , the slides were stained with hematoxylin, dried, and sealed with neutral resin. Detailed steps of immunohistochemistry are provided in the Ser129α-Syn. In a high power field of view, starting from one end of the myenteric plexus of the specimen, we obtained the visual fields of the specimen covering the entire myenteric plexus without overlap. Five visual fields across the plexus were assigned at random for counting, and the ratio of pSer129α-Syn positive neurons was generated from the mean of the number of pSer129α-Syn positive neurons divided by the total number of neurons across the five inspected fields was calculated.The immunohistochemically-stained sections were imaged using a Nikon eclipse 80 microscope and counted as follows: 1. The NF positive neuron count. The number of positive neurons in the myenteric plexus were counted at high magnification (20×) and the NF statistic was determined by the number of neurons observed and divided by the number of visual fields observed; 2. α-Syn expression. A semi-quantitative counting method was used to record the frequency of α-Syn expression, as 0- absence, 1- sparse (≤ 1/4 positive staining area), and 2- small amount (≤ 2/4 positive staining area), 3- common (≤ 3/4 positive staining area), 4- rich (> 3/4 positive staining area) according to criteria from a previous study ; right colon group (Rc); left colon group (Lc), including the descending colon and sigmoid colon; and the rectum group (Re). This was done to exclude the influence of different sampling sites and to verify the rostro caudal gradient.t-test was used for multiple comparisons, including the inter-group comparison of NF and pSer129α-Syn. For semi-quantitative analysis, the Kruskal–Wallis test was used to compare multiple samples between groups. The correlations were analyzed using Pearson correlation, except for the correlation analysis involving α-Syn and tumor staging, which was analyzed using the Spearman correlation test. The statistical significance was set at *P < 0.05, **P < 0.01, and ***P < 0.001).All experiments were performed in triplicate. Data are expressed as the mean ± SD. Data normality was tested using the Kolmogorov-Smirnov test. Differences between means were evaluated using a one-way analysis of variance (ANOVA) test followed by a homogeneity test using IBM SPSS Statistics 20 software . A least significant difference (LSD)-A total of 11 male and 8 female patients, aged between 39 and 84 years old, consented to participate and were enrolled in this study. They underwent colorectal surgery for either malignant tumors of the intestinal tract or benign tumors and were not suitable for endoscopic resection. The basic demographic data of patients in this study are shown in p = 0.822). There were inter-group differences in the NF counts among the Rc, Lc, and Re groups  (p = 0.281) . However, neurons in the mucosa and submucosa were too scattered to count and rate. The myenteric plexus was selected for comparison among the groups. Representative immunostaining of NFs in the myenteric plexus is shown in = 0.017) . The neu= 0.281) .p = 0.812) (p = 0.554)   . Figure = 0.812) . The dep= 0.554) , whereas= 0.008) . Semi-quS129α-Syn was observed only in the submucosa and the myenteric plexus and was detected in the cytoplasm of neurons. We used serial section staining to analyze co-localization. The representative distribution and expression of pathological α-Syn were observed, as shown in S129α-Syn deposits. No significant difference was observed in the deposition of pS129α-Syn between groups    (The deposition of p= 0.720) . Correla= 0.004) , suggest= 0.958) .p = 0.751). Spearman correlation analysis suggested a negative correlation between the tumor stage and the NF counts in the myenteric plexus of the ENS  (p = 0.095)   . There w= 0.095) . Further= 0.566) . Taken tSer129α-Syn were detected within the submucosal and myenteric plexus, as well as in enteric nerve fibers. This evidence argues against the role of ENS immunostaining of α-Syn as a diagnostic hallmark for α-Synucleinopathies and related neurodegenerative disorders. Our findings indicated that the level of pSer129α-Syn was age-dependent in the myenteric plexus, suggesting that the accumulation of α-Syn in the ENS might be a physiological change.In the present study, we explored the distribution of α-Syn and its phosphorylation modification in the non-neurodegenerative intestinal tracts. We employed full-thickness normal intestinal tissues from 19 patients with non-neurodegenerative diseases to explore the levels of α-Syn and phosphorylated α-Syn via immunohistochemistry staining. We found that in the intestinal tissues of patients without neurodegeneration, accumulation of α-Syn and pSer129α-Syn were present in individuals with and without PD . It is also supported the hypothesis that the accumulation of α-Syn is a physiological phenomenon.Our study is consistent with previous immunohistochemical studies on intestinal biopsies, in which α-Syn pathology in the ENS is not a proper indicator of α-Synucleinopathies. The presence of α-Syn in the human ENS appears to be a normal finding. In contrast to native α-Syn immunoreactivity, which remained uniform regardless of the patients' age, we found that the level of pSer129α-Syn occurs in the adult brain physiologically  between non-neurodegenerative cases and neurodegenerative cases might also cause different results. However, in the current study, we only focused on exploring the α-Syn status in the nearly normal ENS. Further studies involving larger samples and patients with neurodegeneration patients are required to provide stronger evidence for our conclusions.Ser129α-Syn was observed in the submucosa and myenteric plexus. This finding weakens the basis and reliability of gastrointestinal pathology as an auxiliary diagnosis for α-Synucleinopathies, including PD.Overall, we found that deposits of α-Syn in the ENS were presented in people with non-neurodegenerative disorders in a physiological state. In particular, the age-dependent appearance of pThe original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by Ethics Committee of Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou, China. The patients/participants provided their written informed consent to participate in this study.E-XT, Y-RL, and X-NJ designed the experiments and critically revised the manuscript. K-XH and D-ZZ performed the experiments. L-LB and D-YL collected patient information and specimens. L-LB, YC, and X-NJ analyzed and interpreted the data. L-LB and X-NJ wrote the manuscript. All authors read and approved the final manuscript. All authors contributed to the article 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.


Alpha-synuclein (α-syn) aggregation is the hallmark pathological lesion in brains of patients with Parkinson’s disease (PD) and related neurological disorders characterized as synucleinopathies. Accumulating evidence now indicates that α-syn deposition is also present within the gut and other peripheral organs outside the central nervous system (CNS). In the current study, we demonstrate for the first time that α-syn pathology also accumulates within the liver, the main organ responsible for substance clearance and detoxification. We further demonstrate that cultured human hepatocytes readily internalize oligomeric α-syn assemblies mediated, at least in part, by the gap junction protein connexin-32 (Cx32). Moreover, we identified a time-dependent accumulation of α-syn within the liver of three different transgenic (tg) mouse models expressing human α-syn under CNS-specific promoters, despite the lack of α-syn mRNA expression within the liver. Such a brain-to-liver transmission route could be further corroborated by detection of α-syn pathology within the liver of wild type mice one month after a single striatal α-syn injection. In contrast to the synucleinopathy models, aged mice modeling AD rarely show any amyloid-beta (Aß) deposition within the liver. In human post-mortem liver tissue, we identified cases with neuropathologically confirmed α-syn pathology containing α-syn within hepatocellular structures to a higher degree (75%) than control subjects without α-syn accumulation in the brain (57%). Our results reveal that α-syn accumulates within the liver and may be derived from the brain or other peripheral sources. Collectively, our findings indicate that the liver may play a role in the clearance and detoxification of pathological proteins in PD and related synucleinopathies.The online version contains supplementary material available at 10.1186/s40478-021-01136-3. SNCA) have been genetically linked to autosomal dominant forms of PD  α‐syn tg and wild type (WT) control mice were isolated [n = 4, tg n = 6) and aged male mice  expressing the human wild type form of α‐syn (Thy-1-L61) were also used for this study. For tg mice modeling MSA (MBP29) [n = 4, tg: n = 4). For App knock-in mice harboring Beyreuther/Iberian mutation (AppNL−F mice) modeling AD, only brain and aged livers were used . All animals were housed in open cages on a 12:12 h reversed dark: light cycle in a temperature‐ and humidity‐controlled room and properly cared by the animal facility. All experiments involving mice were approved by the Local Animal Ethics Committees. The use and care of the animals were conducted in accordance with the EU Directive 2010/63/EU for animal experiments.Male mice used for this study were anesthetized with isoflurane before being transcardially perfused with 0.9% saline. After perfusion, the brain and liver were isolated then fixed in 4% PFA, followed by 70% ethanol before the tissue was paraffin embedded. Samples from young  , only brn = 4) were used for the study. Mice were anesthetized with isoflurane and were stereotactically injected with 5 μg total protein per brain . Material was injected with a Hamilton syringe at a rate of 0.1 μl per min into the right dorsal striatum  at a depth of 2.6 mm below the dura, with the needle in place for 10 min at target. After recovery from surgery, animals were monitored regularly for weight loss and general health status. Animals were sacrificed at 1-month post-protein injection by isoflurane anesthesia followed by transcardial perfusion with 0.9% saline. Brains and livers were then isolated and fixed in 4% PFA, followed by 70% ethanol and subsequent paraffin embedding as described above.The oα-syn preparations were diluted into sterile PBS and sonicated briefly before intracerebral injection. Male C57bl/6 N mice  used for excitation to acquire Z-stacks of consecutive confocal images. Overview images were taken using a Zeiss LSM 780 equipped with 405-, 488-, 559-, and 633 nm laser lines or a LSM 700 (see above) confocal laser-scanning microscope or with a Leica SP5 TCS MP inverted point-scanning confocal equipped with multiphoton for two photon excitations (710–1040 nm) equipped with a digital camera for widefield imaging.t-test when comparing two genotypes or two-way ANOVA with Tukey’s test when comparing multiple samples and genotypes. For Western blot analyses, band intensities were quantified using ImageJ software (Fiji) software, and values with arbitrary units were normalized to the signal obtained from the protein loading control. In each data group, the results are expressed as the mean ± SEM. For semi-quantitative RT-PCR, statistics were calculated using 2−ddCt values, using One-way ANOVA with Tukey analysis for multiple comparisons. All data analyses were performed with GraphPad Prism 7.0 . Findings were regarded as significant when *p < 0.05, ****p < 0.0001.Statistical analyses were performed using a two-tailed unpaired Student’s Given the abundant expression of Cx32 within liver hepatocytes and its recently identified role in promoting oligomeric α-syn uptake, we assessed whether the liver is susceptible to α-syn accumulation in PD. Therefore, we first investigated whether human hepatocytes could take up α-syn protein assemblies associated with PD . Thus, wTo further assess oα-syn uptake in primary human hepatocytes, we immunolabeled oα-syn with a pan-specific human α-syn antibody (14H2L1 (14H)) targeting the C-terminal region of the α-syn molecule (amino acids 121–140)  on Tg brain and liver samples as well as non-Tg tissue sample controls (18 months). Using specific probes targeting human α-syn  under control of the neuronal Thy-1 promoter  antibody which recognizes α-syn pathology in PD brain . Using cWe next investigated the aggregation state of α-syn within the liver using Thioflavin T, a molecular probe known to stain fully mature amyloid structures in multiple neurological conditions. In our hands, however, Thioflavin T failed to label any of the α-syn deposits within aged A30P liver (data not shown), suggesting a limited sensitivity of the probe and/or the lack of fully mature α-syn amyloidogenic structures within the liver. To overcome this technical limitation, we next stained tissue sections with a highly sensitive class of amyloid dyes commonly known as luminescent conjugated oligothiophenes (LCOs), which are ligands identified to stain protein aggregates that are formed early in the aggregation process –40, 55. Given that the emission spectra of LCOs provide clues into the amylogenic state of its target , we nextTo further explore whether α-syn is transported from the brain to the liver, we took advantage of a widely established mouse model of PD involving α-syn injections in the brain leading to α-syn self-assembly and accumulation that is detected within the brain approximately within 20–30 days post-protein injection , 58. ThuGiven the inflammatory response generated by α-syn deposition in the brain and other organs outside the CNS, we next investigated whether the A30P liver is susceptible to inflammation as a result of a progressive α-syn deposition. We therefore performed hematoxylin and eosin (H&E) staining on young (3 months) and aged (18 months) liver tissue sections from WT and A30P mice. In WT mice, we observed no clear signs of inflammation regardless of age Fig. C, D. IndGiven the progressive inflammatory state of the A30P liver compared to livers from wild type mice, we next assessed whether inflammatory cells co-localize with α-syn following its transport to the liver. Thus, we performed confocal image analyses on tissue sections from aged A30P mice and identified the presence of CD45-positve leukocytes Fig. A–D, CD11To further determine whether the accumulation of α-syn in the liver is not specific to the A30P model but rather a general phenomenon of synucleinopathies, we investigated the presence of human α-syn within the liver in a model of synucleinopathy overexpressing normal WT α-syn also under control of the Thy-1 promoter  thus modeling AD [AppNL−F mice (20 months) lacked any detectable age-dependent Aβ accumulation within the liver (data not shown). In mice of advanced age, however (24 months), we observed the presence of rare Aβ inclusions within the liver positive for an array of widely used human specific Aβ antibodies  harboring the Beyreuther/Iberian mutation  with confirmed α-syn deposition in the brain . In a double-blinded setting, we identified the presence of α-syn within the liver that was detected by human-specific α-syn antibodies in several neuropathologically confirmed cases as well as in age-matched control tissues . Although α-syn protein expression in neurons is under control of the same neuronal promoter in both A30P and L61 mice (Thy-1), the distinct degrees of α-syn liver accumulation may be due either to the different forms of human α-syn expressed (A30P vs wild-type) or to the differences in transgenic copies between these models. Thus, suggesting that α-syn deposition within the liver may be dependent on the brain region expressed, protein expression levels, the amount of α-syn deposits in the brain and/or the susceptibility of aggregation of the molecule (mutant vs WT), as well as the cell type in which α-syn is expressed (neurons vs oligodendrocytes) . In contSNCA mutations have been described to cause early onset forms of PD  [Currently five E/G/T/V) , 67. WhiE/G/T/V) . SimilarE/G/T/V) , 69. It E/G/T/V) . HoweverWhile we corroborated brain to liver transport following a single stereotactic injection of α-syn oligomers into the striatum of wild type mice 1-month post-protein injection, the route of delivery from the brain to liver remains unknown for both mice and humans. It is possible that the nodosa ganglia efferent neurons which connect the portal veins and hepatic arteries play a role in this process. Indeed, branches of both the vagal and splanchnic nerves directly innervate the liver via the portal area, and these connections are associated with the portal vein and bile ducts (for review please see ). FurtheAlthough we could identify α-syn pathology in the human liver, we were not able to pinpoint any significant differences on the location, accumulation (fluorescence intensity) or cellular morphology between neuropathologically confirmed cases and controls. Moreover, we could not explain why certain clinically diagnosed PD liver cases with high α-syn burden in the brain  had no detectable α-syn pathology in the liver. However, we cannot rule out the presence of α-syn pathology in other parts of the liver, given the size of the human liver and the random distribution of α-syn pathology. On the other hand, we also observed aged-matched control cases without evidence of neurodegeneration but with α-syn pathology in the liver (8 out of 14). The discrepancy to the findings in the animal models might be explained by the pure, brain-only expression of the α-syn driven by CNS-specific promoters in these models. While in the human cases α-syn could potentially reach the liver both from the brain and the periphery in line with bottom-up transmission hypothesis which indicates that the enteric/gut region is first affected with α-syn pathology, before the pathology progresses to the brain . HoweverIt is worth noting that over a decade ago, Ltic and colleagues reported the presence of multiple endogenous protein α-syn isoforms within the liver, kidney, lung, heart, adrenal gland and testis of both fetal and adult rat as well as human tissue samples . The ideTaken together, while multiple organs outside the CNS in PD cases have been reported to contain α-syn deposits, our study is the first to identify the presence of α-syn within liver hepatocytes in both cellular and animal models of PD as well as in humans. While we could identify α-syn deposition both in PD cases and non-diseased control subjects, the pathology was more common in PD patients. Together with our findings from the cell and animal experiments it is reasonable to conclude that liver α-syn could originate from the brain in the LBD-cases.Although the role of α-syn deposition within the liver both in humans and mice remains unknown, we hypothesize that α-syn is delivered from either the gut/peripheral tissues or brain to the liver before being cleared out of the body, as part of the organ’s detoxification and clearance process. Indeed, we showed that human hepatocytes are capable of lowering the levels of α-syn over time. Moreover, the increase in the inflammatory state observed in aged A30P mouse model suggests that if the capacity of pathological protein clearance is insufficient,accumulated α-syn can lead to liver toxicity. In conclusion, we hypothesize that the liver is involved in the clearance of pathological protein aggregates, which may be a crucial mechanism in the removal of neurotoxic α-syn in PD and related synucleinopathies.Additional file 1: Figure 1. Primary human hepatocytes take up oligomeric α-syn assemblies in vitro. A–F) Confocal image analysis of primary hepatocytes incubated with ATTO-550 labeled oa-syn (red) and then immunolabeled with Cx32 (green) demonstrate the internalization of oα-syn and a partial co-localization between the gap junction protein Cx32 . All cells were counterstained with DAPI (blue). Bars = 10 μm.Additional file 2: Figure 2. Immunoprecipitation of human α-syn oligomers pulls down Cx32. A–D) Immunolabeling on primary human hepatocytes or E–H) HuH-7 cells expressing Cx32 (red) using the human specific 14H antibody show no α-syn reactivity in the absence of α-syn treatment. I) Immunoprecipitation of human α-syn oligomers in HuH-7-Cx32 cells treated with α-syn oligomers pulls down Cx32 whereas untreated cells show no Cx32 pulldown and only the low and heavy chains of the antibody used are shown. J) Confocal image analysis of HuH-7 and HuH-Cx32 cells showing the morphology with or without Cx32 expression. Bars A–H = 20 μm and J= 50 μm.Additional file 3: Figure 3. Cx32 expression in HuH-7 cells promote α-syn uptake. A, B) oα- syn uptake in WT HuH-7 cells (white bars) or HuH-7 cells expressing Cx32 (dotted bars) for different time periods using Western blot analysis. C) Quantification of human α-syn deposits in young (3 months) and aged (18 months) A30P liver tissue sections demonstrates a progressive accumulation α-syn over time. *p<0.05.Additional file 4: Figure 4. Human and mouse α-syn expression is restricted to the brain. A–C) Confocal image analysis of liver tissue sections from aged A30P show no immunoreactivity to α-syn D–F) WT livers in the presence or absence of primary antibody (14H) show no immunoreactivity to mouse α-syn. G) qRT-PCR analysis of human α-syn in A30P brain, liver and normal wild type brains. H) qRT-PCR analysis of endogenous mα-syn in A30P brain, liver and normal wild type brain and liver. n.s.= non-significant. ****p<0.0001, Bars = 20 μm.Additional file 5: Figure 5. Age dependent accumulation of human α-syn deposits within the liver of the L61 model of PD. A–F) Confocal image analysis using the 14H antibody shows the presence of human α-syn deposits (red) in young liver sections (3 months) as small puncta located within the portal tracts and liver parenchyma. Insert within panel (A) shows the deposition of α-syn within the brain of the L61 mice at 3 months of age immunostained with pS129 antibodies (green). G–L) Aged liver tissue sections (12 months) showing the progressive accumulation of human α-syn deposits within the portal tracts and liver parenchyma within the L61 model. Insert within panel (H) shows the deposition of α-syn at 12 months of age immunostained with pS129 antibodies (green). All tissue sections were counterstained with DAPI (blue). Bars = 20 μm.Additional file 6: Figure 6. Identification of human α-syn deposits within the MBP29 liver modeling MSA. A–C) Confocal image analysis using the 14H antibody shows the presence of human α-syn deposits (red) in young liver sections (4 months) as small puncta located within the portal tracts and liver parenchyma. Insert within panel A shows the deposition of α-syn within the brain of the MBP29 mice at 4 months of age immunostained for total α-syn (green). D–F) In some instances, we identified the presence of human α-syn within the sinusoidal region likely surrounding inflammatory cells. G–I) Tissue sections lacking primary antibody (14H) show no human α-syn immunoreactivity. All tissue sections were counterstained with DAPI (blue). Bars = 20 μm.Additional file 7: Figure 7. Human and mouse α-syn expression in MBP29 mice is restricted to the brain. A) qRT-PCR analysis of human α-syn in MBP29 brain, liver as well as non- transgenic (non-Tg) wild type brains and liver samples. n.s.= non-significant. ****p<0.0001.Additional file 8: Figure 8. Identification of amyloid beta (Aß) deposits within the liver of NL-F mice liver modeling AD. A–C) Confocal image analysis of NL-F livers (24-month) using the antibody 6E10. Insert within panel A shows the deposition of α-syn within the brain of the NL-F mice at 24 months of age immune-stained with N82E antibodies (green). D–F) N82E shows the presence of human Aß inclusions (green) which co-localize with the rabbit monoclonal mOC65 antibody (red) within the liver parenchyma. G–I) Tissue sections lacking primary antibody  show no Aß immunoreactivity. Bars = 20 μm.Additional file 9: Figure 9. Accumulation of α-syn within the human liver lacks pS129 immunoreactivity. Confocal image analysis on human liver tissue sections immunolabeled with the 211 human-specific antibody (green) demonstrates the presence of human α-syn deposits within human hepatocytes, portal tracts, and liver parenchyma, however, negative for pS129 immunoreactivity both in PD (A–F) and aged-matched controls (CON) (G–L). Bars = 20 μm.Additional file 10: Table I. Primary antibodies and primers used in this study.Additional file 11: Table II. Description of animal models of neurodegeneration used in this study.Additional file 12: Table III.  Liver characterization of animal models of PD and MSA.Additional file 13: Table IV. Clinical and pathological characterization of human cases included in this study.