Corona virus disease 2019 (COVID-19) increases the risk of cardiovascular occlusive/thrombotic events and is linked to poor outcomes. The underlying pathophysiological processes are complex, and remain poorly understood. To this end, platelets play important roles in regulating the cardiovascular system, including via contributions to coagulation and inflammation. There is ample evidence that circulating platelets are activated in COVID-19 patients, which is a primary driver of the observed thrombotic outcome. However, the comprehensive molecular basis of platelet activation in COVID-19 disease remains elusive, which warrants more investigation. Hence, we employed gene co-expression network analysis combined with pathways enrichment analysis to further investigate the aforementioned issues. Our study revealed three important gene clusters/modules that were closely related to COVID-19. These cluster of genes successfully identify COVID-19 cases, relative to healthy in a separate validation data set using machine learning, thereby validating our findings. Furthermore, enrichment analysis showed that these three modules were mostly related to platelet metabolism, protein translation, mitochondrial activity, and oxidative phosphorylation, as well as regulation of megakaryocyte differentiation, and apoptosis, suggesting a hyperactivation status of platelets in COVID-19. We identified the three hub genes from each of three key modules according to their intramodular connectivity value ranking, namely: COPE, CDC37, CAPNS1, AURKAIP1, LAMTOR2, GABARAP MT-ND1, MT-ND5, and MTRNR2L12. Collectively, our results offer a new and interesting insight into platelet involvement in COVID-19 disease at the molecular level, which might aid in defining new targets for treatment of COVID-19–induced thrombosis.The online version contains supplementary material available at 10.1186/s12920-022-01222-y. The coronavirus SARS-CoV-2 is a highly contagious infection that causes a severe respiratory disease known as COVID-19. This disease that has reached a pandemic level, is impacting tens of millions of people worldwide. In the United States, there are around 78 million reported cases, over 4 million hospital admissions, and 900 thousand deaths as of February 2022 , 6 In adTo obtain a comprehensive insight into the pathogenesis of specific disease states, several computational and research methods have been developed . Some ofAt the same time, machine learning and artificial intelligence are getting extensively used in biology , especiaPrevious studies on the mechanisms of thrombosis in COVID-19 disease have primarily concentrated on specific pathophysiological functions, with relatively fewer studies identifying comprehensive regulatory networks. Therefore, in the present study, WGCNA was used to determine gene networks associated with COVID-19 disease in platelets. PRJNA634489 data set- which contained a total of 15 samples from COVID-19 patients and health controls  was usedRNAseq data is publicly available and were downloaded from BioProject accession #PRJNA634489 . Data co≤ 0.001)  was usedRNA seq data for validation was downloaded from the publicly NCBI SRA repository under accession: #PRJNA736410, analyzed and normalized by following the same steps as first data set.The weighted co-expression network was produced using R package “WCGNA”  as per tCorrelation between module eigengenes and the COVID-19 status was calculated to identify key modules that have significant correlation. The correlation values were displayed within a heatmap. The modules that correlated with COVID-19 most significantly were considered as the key modules. Gene significance (GS) was defined as the correlation between gene expression and the COVID-19 status. Module membership (MM) was defined as the correlation between gene expression and each module’s eigengene, and intramodular connectivity (K.in), which measures how connected a given gene with respect to the genes of a particular module, was also calculated using WGCNA. Subsequently, the correlation between GS and MM as well as GS and k.in were examined to verify module-COVID-19 status associations. The correlation analyses in this study were performed using the Pearson correlation as described in the “WGCNA” package . All modTo validate the results of the above mentioned analysis, multiple classification models  were trained using the key modules of the original data set. Those models were employed to classify the samples of a second data set  of plateThe genes in each key modules were extracted from the network and enrichment analysis was performed to further explore the functions of the respective modules. Targetmine  which isWe used R statistical programming language  version The transcript per million (TPM) gene expression data set were filtered based on variance, and 7119 genes in the 15 samples of ten COVID-19 patients and five healthy controls were used to construct the co-expression network. The results of cluster analysis of the samples are demonstrated in Fig. A. To conTo examine the relation of COVID-19 status with the emerged modules, we built the eigengene adjacency matrix by calculating the correlation of the eigengenes matrix after inserting COVID-19 status to the matrix. The heatmap Fig. B showed To further determine the closest modules to COVID-19 status, we re-clustered the eigengenes using single linkage method with absolute correlation as a distance function; the single linkage clustering algorithm looks for closest pair of modules to form a cluster, then cluster them with the next nearest module progressively until one cluster is formed . As demoExplicitly, the higher the correlation between gene significance of genes in a module and their module membership, the higher its importance. Similarly, when the gene centrality in the network increases in parallel with gene significance, that also is strong evidence that key modules are essential in that trait. The correlations between gene significance and module membership as well as between gene significance and intramodular connectivity show that yellow, black, and magenta modules have the highest correlation values with a substantial difference to the next nearest module (Blue R = 0.61)  high correlation between module eigengene and COVID-19 status, 2) close clustering with COVID-19 status using single linkage with absolute correlation distance, 3) high correlation between genes significance and module membership, and 4) high correlation between gene significance and intramodular connectivity. Together those measures confirm the importance of the key modules in COVID-19 statusThe classification models trained using data from key modules genes showed high performance in terms of high balanced accuracy, sensitivity, specificity, Matthews correlation coefficient, as well as, area under the receiver operating characteristic curve (AUC) Fig. , suggestGenes in the selected key modules were ranked according to the intramodular connectivity and the top 20 genes of each key modules were used to visualize the network of each specific module Fig. . Subsequ2+ in the yellow module. The black module shows enrichment of respiratory electron transport, ATP synthesis by chemiosmotic coupling, heat production by uncoupling proteins, citric acid (TCA) cycle, and respiratory electron transport just to name a few  pathway enrichment analyses were performed on the yellow, black, and magenta modules using Targetmine platform, and the top relevant terms of each category are presented in Fig. A. The pafew Fig. B  to either COVID-19 or healthy using machine learning classifiers. The high accuracy of this classification underscores the importance of these platelet gene clusters in the pathogenesis of COVID-19 disease.2+. Indeed, other studies showed the COVID-19 disease to be associated with platelet activation and increased platelet alpha granule secretion, which are critical in the development of thrombosis seen in those patients [Enrichment analysis indicated that the genes in the yellow and black modules were primarily associated with platelet metabolism, energy, and oxidative phosphorylation. Furthermore, the analysis of the yellow module showed enrichment of a host of platelet functional responses/activities, such as platelet degranulation/secretion and increased platelet response to Capatients , 8. It ipatients .The yellow and black modules show strong enrichment in platelet metabolic processes, which is in agreement with the increase in platelet activation. To this end, previous data have shown that platelet transition from inactive to active state requires alteration in ATP availability , and furOne particular characteristic of platelet apoptotic processes is phosphatidylserine (PS) exposure, which is essential for the generation of thrombin . PS expoAdditionally, we identified hub genes in each of the key modules. For example, in the yellow module the COPE, CDC37, and CAPNS1, which are protein coding genes involved in vesicle-mediated transport, positive regulation of cellular processes, and regulation of interferons. Furthermore, some of these protein coding genes have also been investigated in platelets and shown to regulate important aspects of their function –54, InteThe hub genes of the black module, AURKAIP1, LAMTOR2, and GABARAP are linked to regulation of mitochondrial activity, regulation of signaling processes, and protein targeting. Data on the role of these genes in platelets is limited, thus, further investigation is warranted. It is noteworthy that LAMTOR2 is a known regulator of the MAPK/ERK and mTOR signaling pathways , 57, botIn the magenta module, MT-ND1 , MT-ND5 In addition to the identified hub genes, a number of other canonical platelet genes in the yellow and black modules were also associated with platelet function. For example, SLEB and ITGA2B protein coding genes were present in the yellow module with high intramodular connectivity (ranked in the top 50) and both proteins are critical for platelet function . MoreoveThe present study has certain limitations that should be noted. Firstly, the analysis focused on only one data set, due to limited access to platelet gene expression data that were collected from COVID-19 patients. Therefore, additional data sets should be analyzed, if available, to validate our findings and/or obtain more representative results. Also, the number of samples was 15, which may be associated with some noise, albeit it is the minimum number of samples recommended for co-expression analysis by WGCNA. Finally, any limitations in the original study, from which the data was obtained will also be reflected in the results of this study.In conclusion, our co-expression analysis of a platelet RNAseq data set from COVID-19 patients and healthy controls revealed 16 modules, amongst which the yellow, black, and magenta were identified as the most critical in COVID-19 disease and validated using machine learning. Additionally, nine hub genes were determined to potentially serve key roles in the pathophysiological mechanisms of COVID-19 in the context of platelet biology. The positively associated yellow and black modules were identified to be involved in platelet degranulation, energy metabolism, and mitochondria. The negatively associated magenta module was associated with interactive pathways of apoptosis. These data should help expand our understanding of the underlying mechanisms of thrombosis in COVID-19 disease and help promote and guide future experimental studies to investigate the roles of the protein coding genes in the pathophysiology of this disease. Additionally, these genes may serve as novel therapeutic targets for treating patients.Additional file 1. Figs. S1–S3.Additional file 2. Table S1: WGCNA eigengenes.Additional file 3. Table S2: WGCNA modules.Additional file 4. Table S3: Gene significance.Additional file 5. Table S4: Intramodular connectivity.Additional file 6. Table S5: Hubgenes cross check with Disgenet database.


This is the first report that SCN8A may be associated with CwG. Our report may provides evidence for the genetic etiology of CwG and expands the phenotypic and genetic spectrum of SCN8A-related disorders, which previously included severe developmental and epileptic encephalopathy (DEE) phenotype, benign epilepsy phenotype, spectrum of intermediate epilepsies, and patients with cognitive and/or behavioral disturbances without epilepsy. Phenotype of CwG has a good prognosis, and it does not require long-term antiepileptic therapy. Overtreatment should be avoided clinically. However, the conclusion needs to be further defined by long-term follow-up and similar clinical reports. In spite of this, our clinical observation provides possible evidence for future studies on the relationship between SCN8A and CwG.Benign convulsions with mild gastroenteritis (CwG) is characterized by afebrile convulsions accompanied by mild gastroenteritis, and it can be considered after central nervous system infection, hypoglycemia, electrolyte disturbance, and moderate and severe dehydration are excluded. Previous studies have suggested that genetics may be involved in CWG. Herein, we reported a novel  Benign convulsions with mild gastroenteritis (CwG) was first reported by Morooka in 1982 in Japan . Most ofSCN1A, SCN1B, and PRRT2 genes in CwG patients, but no positive results were found (SPTAN1 gene by next generation sequencing (NGS) in a family, including a 13-year-old sister, an 8-year-old brother and their 39-year-old mother  C>G in SCN8A gene that was not detected in either parent. The variant led to protein changes p.Pro1835Ala (NM_014191.4). The variant (p.Pro1835Ala) was a de novo heterozygous variant (PS2), which affected highly conserved amino acid region. It was not found in the normal control population in multiple databases, such as gnomAD, ExAC, 1,000 Genomes, ESP6500 (PM2_Supporting). Various statistical methods predicted that the variation would have deleterious effects on gene products (PP3), such as SIFT (Damaging), MutationTaster (Disease_causing), FATHMM (Damaging), PROVEAN (Damaging), DANN (Damaging), CADD (Damaging) and Eigen (Damaging). In addition to this, the missense mutation of SCN8A gene had a high pathogenic possibility, and the Z-score was 7.64 in gnomAD database (PP2). Hence, the variant was classified as likely pathogenic in accordance with the ACMG guideline (PS2 + PM2_Supporting + PP2 + PP3).The patient was a 36 months old boy. The Chinese boy was born full term with normal birth weight. She had no history of transient hypoxia at birth. There was no family history of neurologic disorders. He developed acute gastroenteritis symptoms at 19 months, presenting with 3 times of vomiting and diarrhea (3 times daily). A day later, the boy began with 3 generalized tonic-clonic seizures (GTCS) without fever in 1 day. The duration of each convulsion was 2–3 min. Since then, phenobarbital was given . However, he still had two more similar convulsions. After treatment of diazepam , there was no further convulsion. Vomiting resolved within 2 days, while diarrhea recurred over 5 days (2–4 times a day) and then resolved. Dehydration did not occur throughout the course of the disease. In the interictal phase, the child's mental state was normal and his consciousness was clear. However, 10 days later, he developed symptoms of gastroenteritis again, including diarrhea and vomiting, and had one GTCS a day later. The duration of the convulsion was 2 min. The symptoms of the mild gastroenteritis lasted for 4 days, and the convulsions only occurred once during the course of the gastroenteritis. We did not give the child long-term antiepileptic treatment. After a long-term follow-up of 17 months, he never had another seizure. The child's motor and intellectual milestones were the same as those of normal children. Physical examination at the age of 19 months showed a height of 83 cm (50–85th centile), weight of 13.5 kg (85–97th centile) and head circumference of 47 cm (25–50th centile). His consciousness was clear. Cardiac auscultation was normal. Muscle strength and tone were normal. Knee reflex were normal and babinski signs were negative. EEG showed slow background activity during wakefulness in interictal periods, and no epileptic discharges were observed . RotavirIn our case report, the patient presented with multiple afebrile convulsions accompanied by mild gastroenteritis, and he did not present with electrolyte disturbances, dehydration, hypoglycemia, or central nervous system infection. In addition, he did not receive long-term antiepileptic medication. After a long-term follow-up of 17 months, he did not develop convulsions again, and his motor and cognitive development was normal. he has a good prognosis. In the first course of acute gastroenteritis, vomiting resolved within 2 days, while diarrhea resolved within 5 days. Ten days later, he developed symptoms of gastroenteritis again, including diarrhea and vomiting. The symptoms of the mild gastroenteritis lasted for 4 days. The convulsions only occurred during the course of the gastroenteritis. During a 10-day period, there were no symptoms of gastroenteritis or convulsions. Therefore, I think it was two episodes. Recent study has shown that children with CwG have a possibility of recurrence, with a recurrence rate of about 6.3%   pathogenic mechanism, which cause ultimately hyperactivity of the ion channel. On the contrary, variants causing loss of function (LoF) are related with patients with cognitive and/or behavioral disturbances without epilepsy, Such as ID, ASD, myoclonus, and ataxia  . In recehy (DEE) –17. Mostd ataxia . HoweverSCN8A gene might be involved in pathogenesis of CwG. Phenotype of CwG has a good prognosis. It does not require long-term antiepileptic therapy, and overtreatment should be avoided clinically. However, the conclusion needs to be further defined by long-term follow-up and similar clinical reports. In spite of this, our clinical observation provides possible evidence for future studies on the relationship between SCN8A and CwG.In conclusion, our clinical observation suggests that variants in The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the Research Ethics Committee of Children's Hospital of Jiangxi Province. Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin. Written informed consent was obtained from the individual(s), and minor(s)' legal guardian/next of kin, for the publication of any potentially identifiable images or data included in this article.All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The nutritional adequacy of the meals for children aged from 6 to 11 years was estimated using the mean adequacy ratio  as the mean percentage of daily recommended intake for 23 nutrients and the mean excess ratio  as the mean percentage of excess compared to the maximum daily recommended value for three nutrients. This analysis of actual school meals shows that both vegetarian and non-vegetarian meals had a similar good nutritional quality with MAR/2,000 kcal of 87.5% (SD 5.8) for vegetarian and of 88.5% (SD 4.5) for non-vegetarian meals, and a MER/2,000 kcal of 19.3% (SD 15.0) for vegetarian and of 19.1% (SD 18.6) for non-vegetarian meals. GHGE were more than twofold reduced in vegetarian compared to non-vegetarian meals (0.9 (SD 0.3) vs. 2.1 (SD 1.0) kgC02 eq/meal). Thus, increasing the frequency of vegetarian meals, by serving egg-based, dairy-based or vegan recipes more frequently, would reduce GHGE while maintaining adequate nutritional quality of primary school meals.Since 2018 in France, national regulation mandates that school canteens serve a weekly vegetarian meal to reduce school canteens' environmental impact in addition to previous regulations imposing nutritional composition guidelines. However, a lunch without meat is often perceived as inadequate to cover the nutritional needs of children. The present study aims to assess the nutritional quality and greenhouse gas emissions (GHGE) of vegetarian and non-vegetarian school meals served in primary schools in Dijon, France. The catering department provided the composition of 249 meals served in 2019. Nutritional content and GHGE were retrieved from national food databases. The portion size of each meal component was the standard portion size recommended by the relevant French authority (GEMRCN). Meals were classified into vegetarian meals, i.e., without meat or fish ( Non-communicable diseases  as well as environmental threats  require identifying dietary changes that will improve nutritional quality and reduce the environmental impact of diets , 2. Becan = 41 among the 800 dish options).Currently, in France, 8.5 million children aged 3 to 17 years eat at least once a week at school canteens. Among children aged 3 to 10 years, 58% eat lunch regularly at the school canteens, i.e., at least 4 days a week . In FranIn 2018, a law for the “balance of trade relations in the agricultural and food sector and sustainable healthy food accessible to all”  was adopted in France 3. This lReduction of meat consumption in school canteens is key to building more environmentally friendly food systems, but evidence based on the analysis of school meals from real life is still needed to convince catering actors, parents and children of the nutritional quality of vegetarian meals. The challenge is thus to demonstrate that a high level of nutritional quality could be maintained while limiting the negative impacts of meals on the environment and, in particular, global warming.In the present study, we used validated indicators to compare the nutritional quality and environmental impact of all meals (66 vegetarian and 183 non-vegetarian) that were served in 2019 to children aged from 6 to 11 years old in primary schools in Dijon, France. We hypothesized that vegetarian and non-vegetarian meals would have similarly good nutritional quality, whereas the environmental impact would be significantly reduced for vegetarian meals compared to non-vegetarian meals. Moreover, we aimed at analyzing more closely the nutritional quality and environmental impact of school meals based on the type of protein dish.n = 249). In this context, school meals delivery is organized by the municipality service. The central kitchen of Dijon produces and serves 4,000 meals every day to children aged 6 to 11 years  distributed among the 38 primary school canteens  to children in primary schools in Dijon, France n = 9. In thihttps://osf.io/fk7cq/). The meal database contained the 249 meals served in 2019 corresponding to five meals served per week (excluding weekend) during the school period and school holidays. A meal was composed of five or four components, among a starter, protein dish, side dish , dairy product, and dessert, with a 30- or 40-g portion of bread. Meals were split into two main categories: vegetarian  and non-vegetarian (n = 183).Three databases were created from the collected information: the meal database, the dish database and the recipe database  or non-vegetarian. Moreover, the meals were categorized based on the subcategory of the protein dishes. Five different subcategories were defined based on their level of GHGE , from thThe 433 food items were included in the recipe database and identified by a name and a code. The portion size of each food item (in grams) was established by the central kitchen and closely related to the GEMRCN recommendations for primary school children . All foo2 equivalent) of each food item was based on the AGRIBALYSE v3.0 table providing reference data on the environmental impacts of agricultural and food products obtained through life cycle analysis, comprising the following contributions to GHGE: agriculture, transport and packaging , the best-known and most-used climate change indicator  and then summed at the dish level and at the meal level.To estimate the overall nutritional quality of the meal, we used the mean adequacy ratio (MAR) which estimates the average content of several nutrients expressed as a percentage of recommended intakes . In the The MAR/2,000 kcal is reported on a scale from 0 to 100%, where 100% indicates that the daily requirements for all the nutrients were met. Each ratio was truncated to 1 so that a large quantity of one nutrient could not compensate for a small quantity of another, hence nutrient coverage beyond children's daily needs was not considered.We also calculated the mean excess ratio (MER) which estimates the excess compared to the daily maximum recommended values (MRV) of three nutrients that should be limited: saturated fatty acids (SFA), salt and total sugars. MER was expressed as the percentage of excess compared to the MRV for 2,000 kcal of a meal, as indicated in Equation (4) . The MRVThe MER/2,000 kcal is reported in percentage of excess, where 0% indicates that none of the three nutrient limits were reached. Each ratio was limited to 1, so that a small quantity of one nutrient could not compensate for a large quantity of another.t-tests were performed on weight, energy, GHGE, MAR/2,000 kcal, MER/2,000 kcal and contents in 26 nutrients per 2,000 kcal of a meal. For each nutrient and each type of meal, we also compared the percentage of RDI or MRV for 2,000 kcal of meal to the target value (100%) using one-sample Student's t-tests. Then, we performed ANOVA models to compare outcome variables  between the five subcategories of meals based on their protein dish  and pairwise post hoc comparisons were performed.Statistical analyses were conducted after computation of the data at the meal level, as explained above. Correlations between total weight of meals, energy content, GHGE, MAR/2,000 kcal and MER/2,000 kcal were computed with Pearson correlation coefficients. To compare vegetarian meals and non-vegetarian meals, two-sample Student's Statistical analyses were performed with SAS software version 9.4. The level of significance was set to 0.05 for all of the analyses and Bonferroni correction was used to control for multiple testing across the 26 nutrients .2 eq (SD 1.0). The average MAR/2,000 kcal was 88.3% (SD 4.9), indicating that on average 88% of the RDI for the 23 nutrients would be covered by 2,000 kcal of a school meal. The average MER/2,000 kcal was 19.1% (SD 17.7) indicating that on average the MRV for three nutrients  would be exceeded by 19% with 2,000 kcal of a school meal.Data from 249 meals were considered for the present analysis. The average weight of a meal was 464 g (SD 64), and the average energy content was 659 kcal (SD 125) representing 33% of the recommended daily energy intake for children aged 6 to 11 years old . The average greenhouse gas emissions (GHGE) of a meal was 1.8 kgCOPearson's correlations showed a positive association between weight and energy content and a negative association between weight and MER/2,000 kcal highlighting that the content in nutrients to limit was lower in school meals of higher weight. There was also a negative association between energy content and MAR/2,000 kcal indicating that school meals of better nutritional quality were also those of lower energy content. No association was found between GHGE and weight, energy content nor nutritional quality of the meals .n = 249), non-vegetarian (n = 183) and vegetarian meals (n = 66) are shown in 2eq (SD 0.3), whereas a non-vegetarian meal emitted 2.1 kgCO2eq (SD 1.0). MAR/2,000 kcal for non-vegetarian and vegetarian meals was not significantly different, likewise for MER/2,000 kcal.Among the 249 meals served in 2019 in Dijon school canteens, 183 were non-vegetarian (73.5%), and 66 were vegetarian (26.5%), which is greater than the recommended frequency of one per week  established by the EGalim law. The nutritional quality and environmental impact of all meals  in both vegetarian and non-vegetarian meals: proteins, fibers, vitamins B1, B2, B3, B6, B9, B12, E, A, potassium, iron, magnesium, zinc, copper, iodine, selenium, linoleic acid (LA) and docosahexaenoic acid (DHA) . In contn = 56); pork and poultry (n = 68); fish (n = 55); eggs and/or cheese (n = 40); and vegan (n = 30) are shown in F = 1.01, p = 0.404] despite different weights . Meals with vegan or fish-based main dishes had a significantly higher weight than meals with beef, veal or lamb dishes. A significant effect of the meal subcategory on GHGE , MAR/2,000 kcal  and MER/2,000 kcal  was noted. GHGE differed between all five subcategories: meals with beef-, veal- or lamb-based dishes emitted the most GHGE followed by meals with fish-based dishes, meals with pork- or poultry-based dishes, meals with eggs and/or cheese-based dishes, and finally vegan dishes, with an approximately five-fold reduction compared to ruminant meat-based meals. MAR/2,000 kcal was significantly lower for meals with vegan dishes compared to meals with eggs and/or cheese or fish dishes, but the difference was small . MER/2,000 kcal was significantly lower for meals with vegan dishes compared to fish-based meals and meals with eggs and/or cheese dishes. Only meals with vegan dishes had SFA contents below the MRV [80.5% (SD 33.0)]. Each subcategory of meals had some nutrients below the RDI or above the MRV. Nutrients in deficit (significantly below RDI) and nutrients in excess (significantly above MRV) in the five meal subcategories are presented in The nutritional quality and environmental impact across the five meal subcategories based on protein dish: beef, veal, lamb  to present the net intake per meal. These tables are available in 2eq) were consistent with results found in previous studies: 1.7 kgCO2eq  kcal. This result is similar to values found in previous studies investigating the nutritional quality of school meals in France: 712 kcal  and 590 Our data provide specific insight into how the nutritional needs of children are covered in a given school canteen organization. Based on the consumption of 7- to 10-year-old children from the French INCA3 study, some nutrients  have been identified as not reaching the recommendations defined by EFSA . Based oSome nutrients were found in low quantities in school meals compared to the nutritional intake recommendations for children, but others excessively covered dietary needs. In particular, the protein content was high for vegetarian meals , even for meals with vegan-dishes based meals (260.7%); not to mention the skyrocketing rate in non-vegetarian meals (400.1%). Thus, based on our data, most school lunches cover daily protein needs in excess (>100%).No associations were found between GHGE and the weight or energy content of the meals. This result differs from those of a study examining the relationship between the environmental impact and nutrient content of sandwiches and drinks sold in a university canteen (UK), showing that the environmental impact score was positively associated with portion sizes and calories . This diNor the correlation between GHGE and MAR/2,000 kcal, neither between GHGE and MER/2,000 kcal were significant. We showed that non-vegetarian meals emitted more GHGE than vegetarian meals and that the nutritional quality of non-vegetarian and vegetarian meals was similar, which may partly explain the lack of association between GHGE and indicators of nutritional quality. In a context where reducing GHGE from the food system is necessary and urgent to meet the Paris Agreement's goal of limiting the increase in global temperature to 1.5 or 2°C , we highThe present study has several limitations. First, we used nutritional and environmental indicators from CIQUAL, CALNUT and AGRIBALYSE databases that include values on average food items. We evaluated meal environmental impact based on GHGE but we did not use other indicators such as eutrophication, acidification, toxicity, biodiversity which may encompass other important impacts on our planet. Moreover, one must note that AGRIBALYSE database did not provide a distinction between organic and conventional food items, although their impacts may differ. For nutritional evaluation, we did not consider actual food items composition but average food items composition from the French reference databases. Nutrient content may vary especially for salt and fat depending on culinary practices. As the dietician from the central kitchen reported trying to limit the salt and fat content of the meals, the MER may have been overestimated for some recipes. The confidence levels for pairing with average food items were mostly high. For 92 items out of 433 with the lowest confidence levels, it would have been interesting to retrieve actual food items composition to estimate nutrient content more precisely.The bioavailability of nutrients was not considered in the nutritional evaluation. The bioavailability of iron and zinc has been showed to be lower when they come from plant-based products . BioavaiAnother limitation is that we considered the food portions used by the central kitchen to estimate production needs (closely related to the GEMRCN recommendations) to calculate the nutritional quality indicators. However, the portions established by the central kitchen were probably different from the portions actually served to children and different from the portions actually consumed by children. Therefore, it would be needed to evaluate the portions of each component consumed by the children to estimate the variability in coverage of individual nutritional needs. This could be achieved by using individual food intake measure methods used in previous studies in children , 47. ThiThis study has several strengths. First, it was based on a unique database of meals served in one primary school canteen system during a one-year period. For the first time, the nutritional quality and GHGE were conjointly estimated for actual meals within one system. Based on the availability of yearly data, further studies could examine the evolution of nutritional quality and GHGE over several periods or seasons throughout the year or over several years. More globally, in the context of a current initiative of the Dijon catering system toward more sustainable food systems, these data provide a baseline estimation to follow the evolution of Dijon school catering during the coming years. Moreover, integrating other indicators reflecting other dimensions of sustainable food systems  such as In this study, we assessed the nutritional quality and GHGE of meals served in 2019 in Dijon primary school canteens. We showed that all meals were of good nutritional quality, notably as 2,000 kcal of a meal could cover on average 88% of the recommended daily needs for 23 nutrients. Vegetarian meals  had on average two-fold lower GHGE compared with non-vegetarian meals. Thus, increasing the frequency of vegetarian meals beyond the current regulation (one per week) seems to be a good strategy to meet the double challenge of maintaining good nutritional quality and reducing the carbon impact of school catering. The school catering system of Dijon, like other municipalities, could integrate more vegetarian meals by increasing the frequency of dishes based on eggs, dairy products or vegan recipes. Future research work aimed at improving the sustainability of school catering, which would be nutritionally adequate for children and respectful of the environment, should ensure that children's habits and tastes, costs for families and for farmers are also taken into account.https://osf.io/fk7cq/.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: Conceptualization, methodology, and writing—review and editing: JD, LM, and SN. Formal analysis and writing—original draft preparation: JD. Resources: J-MG. Data curation: JD and LM. Supervision: LM and SN. Funding acquisition: SN. All authors contributed to the article and approved the submitted version.This research was funded by a grant to SN , a grant from the Dijon Municipality in the frame of the Dijon Alimentation Durable 2030 initiative (grant #00006226-29dmact11), which supported the PhD fellowship of JD, and a grant of the Caisse des dépôts et consignations through the Territoires d'innovation funding scheme (grant #00005065). Funders were not involved in the data analysis plan.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Enchytraeus crypticus (oligochaeta), was exposed through contaminated soil with nanopolystyrene (representative of nanoplastics (NPls)), alone and in combination with diphenhydramine , silver nitrate  and vanadium nanoparticles . AgNO3 and VNPs decreased E. crypticus reproduction at 50 mg/kg, regardless of the presence of NPls. Moreover, at the same concentration, both single and combined VNP exposures decreased the E. crypticus survival. On the other hand, DPH and NPls individually caused no effect on organisms’ survival and reproduction. However, the combination of DPH (10 and 50 mg/kg) with 300 mg NPls/kg induced a decrease in reproduction, showing a relevant interaction between the two contaminants (synergism). Our findings indicate that the NPls can play a role as vectors for other contaminants and can potentiate the effects of pharmaceuticals, such as DPH, even at low and sub-lethal concentrations, highlighting the negative impact of mixtures of contaminants (including NPls) on soil systems.Plastics in all shapes and sizes have become widespread across ecosystems due to intense anthropogenic use. As such, they can interact with other contaminants that accumulate in the terrestrial environment, such as pharmaceuticals, metals or nanomaterials (NMs). These interactions can potentiate combined toxic effects in the exposed soil organisms, with hazardous long-term consequences to the full ecosystem. In the present study, a terrestrial model species,  Industrial development over the past 70 years has brought innovative ideas into daily life, such as plastic-based durable household appliances, as well as nano-based electronics and other applications ,2. HowevPlastic can be degraded into micro  and nano size , as a result of mechanical and chemical processes within the soil matrix . Due to Enchytraeus crypticus where real plastic products were tested: a combination of plastics  containing NMs (copper oxide (CuO), iron(III) oxide (Fe2O3), organic pigment and multi-walled carbon nanotubes (MWCNT)) [Currently, the majority of ecotoxicological studies focus on exposures of one contaminant alone, underestimating the effects of contaminant mixtures that are occurring in soil due to anthropogenic activities. Few studies have been published on the effects on soil organisms due to co-contamination by MPls and other toxicants ,15,16. H(MWCNT)) . This is(MWCNT)) .E. crypticus (oligochaeta) [3, metal) and vanadium nanoparticles . In prior studies, these contaminants have been shown to be toxic to different soil model organisms [In order to mitigate these knowledge gaps, the focus of the present study was the exposure of the commonly used standard terrestrial model invertebrate ochaeta)  to the crganisms ,20,21. Trganisms , and therganisms  makes thE. crypticus, compared to the single exposures. This work provides novel information on the role of NPls in the hazardous potential of known contaminants to relevant soil model organisms such as enchytraeids, mimicking more realistic scenarios that already occur in the field, with potential long-term effects on ecosystem composition. Enchytraeids live in the litter layer and the upper mineral soil, feeding on fungal hyphae, microorganisms and dead organic matter [E. crypticus to test the effects of different anthropogenic contaminants  [In the present study, it is hypothesized that in a co-contamination scenario, the interaction between NPls and each contaminant will result in a higher level of toxic effects, in particular in reproduction of c matter . Moreovec matter . Previouand NMs) ,11,25.E. crypticus , Westheide and Graefe, 1992, was used for the tests. According to Directive 2010/63/EU of the European Parliament and of the Council of 22/9/2010, invertebrates such as E. crypticus are permitted biological models for scientific experimentation and are free from Ethical Statements. The cultures were kept in agar, consisting of Bacti-Agar medium  and a sterilized mixture of four salt solutions as previously described [3)) was acquired from Bangs Laboratories, Inc. According to the supplier, NPls had mean diameter of 44 nm and a surface area of 1.299 × 1014 µm2/g. NPls dispersion was centrifuged prior to the ecotoxicity tests using a Vivaspin® 2 mL ultrafiltration device  to remove SDS and NaN3. Then, the NPls stock dispersion was characterized by hydrodynamic size , evaluated by dynamic light scattering  and by zeta potential (ZP), assessed by electrophoretic light scattering  to verify the characteristics given by the supplier. The Zetasizer Nano ZS  also allowed to obtain the polydispersity index (PdI) of the NPls dispersion. DPH hydrochloride (powder) was acquired from Sigma-Aldrich, with a molecular weight of 291.82 g/mol and a purity of 98% and an n-octanol/water partition coefficient (logKow) of 3.27. AgNO3 (powder) was acquired from PanReac, with a molecular weight of 169.87 g/mol and 99.8% purity. VNPs dispersion (2% in triton X-100 and water) was purchased from Nanoshel UK Limited , presenting an average particle size (APS) between 80 and 100 nm and a purity of 99.9%. As for NPls, VNPs stock and test dispersions were characterized by HS and ZP.Nanopolystyrene dispersion (10.06% solids in deionized water with 0.1% sodium dodecyl sulfate (SDS) and 0.05% sodium azide (NaN3 and VNPs), HS, ZP and PdI of the test dispersions—NPls single exposures (1.5 and 300 mg/kg) combined with DPH, AgNO3 and VNPs (10 and 50 mg/kg)—were measured at day 0 of the ecotoxicity tests. To investigate the interaction between NPls and the other contaminants  was used for the tests and had the following main characteristics: pH = 5.8, organic carbon = 1.71%, cation exchange capacity = 9.2 meq/100 g, maximum water-holding capacity (WHC) = 44.8% and grain size distribution of 8.9% clay, 13.9% silt and 77.2% sand.3 test solutions, they were dissolved in ultrapure water, no organic solvent needed. The required volumes of NPls test dispersions prepared in ultrapure water—single and combined with DPH, AgNO3 or VNPs—were added to the pre-moistened soil (in which water was added before) until 50% of the WHC maximum and mixed manually. Moreover, for the single exposures, test solutions of DPH and AgNO3 and test dispersions of VNPs, prepared in ultrapure water, were added to the soil, as described for NPls test dispersions. The replicates were mixed individually as recommended by the Organisation for Economic Cooperation and Development (OECD) guidelines [3 and VNPs (10 and 50 mg/kg), as well as the binary mixtures of NPls with each co-contaminant . The selected single VNPs, AgNO3 and NPls concentrations, the toxicities of which were previously studied [E. crypticus were not known yet (single and combined with NPls). All the ecotoxicity tests started 1 day after soil spiking, similar to other previous published studies involving the testing of nano-based materials [The soil was dried  before use. The control soil was prepared by adding deionized water to adjust to the adequate moisture content (50% of the maximum WHC). Due to the presence of 2% of triton X-100 on the VNPs stock dispersion, a solvent control was also performed, adding the same volume of triton X-100 present in the highest tested concentration of VNPs (50 mg/Kg) (corresponding to 0.2% of triton X-100). For DPH and AgNOidelines . Soil sp studied ,25, wereaterials ,27,28,29E. crypticus was an adaptation to the OECD guideline 220, allowing their development  in the contaminated media . Hence, the used time of exposure was 28 days (instead of 21 days). The test ran at 20 ± 1 °C and a 16 h: 8 h (light: dark) photoperiod. During the test, food (11 mg) and water content (based on weight loss) were replenished weekly. Four replicates per experimental condition (n = 4) were used. An additional replicate per condition (without organisms) was prepared to measure the pH values.The ERT procedures followed the OECD guideline 220 , with adAt the end of the test period, the organisms were fixed with ethanol and stained with 1% Bengal rose in ethanol (minimum of 4 hours). Soil samples were sieved through meshes with decreasing pore size  to separate the organisms from most of the soil and facilitate counting. Adult and juvenile enchytraeids were counted using a stereomicroscope, and survival (number of adults) and reproduction (number of juveniles) were evaluated.t-test.Graphics and statistical analysis were performed applying the Sigma Plot 14.0 software package . Shapiro–Wilk and Levene’s tests were applied to evaluate the normality and homoscedasticity of data, respectively. To assess differences between control and treatments, one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparison post hoc test were employed. When data did not follow a normal distribution, a non-parametric Kruskal–Wallis test was performed. Differences between control and solvent control were carried out using a Student’s 3 and VNPs) to the organisms’ survival and reproduction response, a two-way ANOVA was performed, followed by post hoc Dunnett’s test. To assess the contribution of the interactions between NPls and the other contaminants  presented the expected HS with lower PdI , being she metal .E. crypticus survival and reproduction after 28 days of exposure to soil contaminated with NPls, single and combined with DPH, AgNO3 or VNPs, are shown in The results of E. crypticus reproduction and survival between the control and 1.5 or 300 mg NPls/kg  combined with 10 or 50 mg DPH/kg decreased the 3 (50 mg/kg) decreased the E. crypticus reproduction, compared to control  on the organisms’ reproduction was similar to the one observed in the correspondent single AgNO3 exposure  with the highest tested concentration of AgNOE. crypticus survival compared to control and the respective single NPls exposures  and 50 mg VNPs/kg decreased the E. crypticus and polystyrene-based NPls [w/v) NPls (0.05–0.1 μm particle size) resulted in a significant decrease in E. crypticus cocoon production after 7 days [Despite the few studies available involving sed NPls ,30, one sed NPls . The preCeriodaphnia dubia [Daphnia magna reproduction was affected at concentrations above 0.8 µg/L [E. crypticus after 28 days. However, as DPH is considered persistent in soil [Currently, there is no information on the effects of DPH in survival and reproduction of soil organisms, and limited information on aquatic organisms ,32. Acut0.8 µg/L . The pre in soil  and can  in soil  and exis in soil , it can 3/kg was expected. The tested concentration is within the confidence interval of the 10% effect concentration (EC10) estimated by Gomes et al. [20) and 50% EC (EC50) estimated by Bicho et al. [E. crypticus. As no effects were observed for E. crypticus survival, it is possible that AgNO3 may delay the hatching process and embryotoxicity via blocking of Ca channels, as previously proposed [In the present study, the decrease in organisms’ reproduction observed after the exposure to 50 mg AgNOs et al.  and betwo et al.  for E. cproposed ,36.2) NPs have been shown to produce effects at the minimal tested concentrations (2.5 µg/mL) in human lung cells as well as Gram positive bacteria, related to an increased production of reactive oxygen species (ROS) [E. crypticus reproduction (28 days EC50 = 11.0 ± 1.5 mg/kg) as well as survival [Prior studies on the effects of VNPs on biological systems are in limited number ,37,38. Hsurvival . The finE. crypticus reproduction when compared with the single exposures (where no effect was observed). Furthermore, the significant interaction observed for NPls and DPH (synergism) indicates that there may be a combined effect of the mixture, namely a potentiation by NPls to DPH toxicity [Co-contamination of NPls and DPH resulted in a higher degree of effect in toxicity . Similartoxicity . Concerntoxicity . In fishtoxicity . Prior stoxicity , suggesttoxicity ,45, whicIn addition, the nature of the pharmaceutical is relevant for the adsorption process, as it is mediated mainly by hydrophobic, Van der Waals and hydrogen bonding, together with the role of environmental pH, as presented by McDougall and prior studies . In the 3, it was found that the predominant driver in the observed toxicity was the presence of AgNO3. No different effects were observed between AgNO3 single and combined exposures, suggesting no interaction effect between the metal and NPls. This can be explained by the absence of a functional group (such as carboxyl (COOH) or amino (NH2)) in the NPls used . If the tested NPls contain a functional group, it could promote the binding of the Ag ions with the NPls, and, consequently, form stable complexes. This complex (NPls + AgNO3) could cause a different toxicity than expected. Indeed, a previous study with the brine shrimp Artemia franciscana involving two types of metal-based salts (potassium dichromate (K2Cr2O7) and copper sulfate (CuSO4)) co-exposed with amino-functionalized polystyrene NPls, found an interaction effect between the metals and NPls [Triticum aestivum L.) to polystyrene NPls and cadmium (Cd), the presence of polystyrene induced a slight alleviation of Cd-induced toxicity [A. franciscana, polystyrene NPls decreased the immobilization rate induced by the single CuSO4 exposure [Eisenia fetida exposure to NPls in arsenic (As) and Cd contaminated soil promoted antioxidant response (glutathione S-transferase (GST) activity and malondialdehyde (MDA) content increased) [2Cr2O7 induced a higher immobilization rate in A. franciscana larvae [3 can be supported by the results from the characterization of NPls, since the HS and ZP NPls values maintained unaltered in the presence of AgNO3. This is likely a result of the dissolution of AgNO3, decreasing the potential for interactions with less hydrophilic NPls.In the combined exposure NPls and AgNOand NPls . In the toxicity . In addiexposure . On the creased) . In the creased) , and thea larvae . The desChlamydomonas reinhardtii, there was no increased uptake of plastic but of silver, promoting toxicity [It was clear that VNPs were the major driver of toxicity, as the combination of NPls with VNPs did not inhibit or increase the VNPs toxicity found in the single exposures. Similar to other metal-based NPs (such as Ag), VNPs can release V ions and cause toxicity . It is ptoxicity . As suchE. crypticus response. The combination which caused the most effects was VNPs and NPls, which caused effects for both tested endpoints: survival and reproduction. However, these effects were due to the VNPs exposure, independent of the presence of NPls. Comparing the results of combined exposures with the correspondent single exposures, different interaction effects, including no interaction and synergism, were found. The presence of NPls did not have an interference (showing no interaction) on the observed effects of VNPs and AgNO3. Yet, the combination of DPH and NPls resulted in toxic effects that were not observed in the single exposures, i.e., there was an interaction effect related to the synergy between both contaminants.The use of three contaminants with distinct nature  in combination with NPls showed clear differences in terms of impact in The complexity of the exposure matrix (soil) provides several challenges to assessing how NPls interact with the surrounding environment and other contaminants . The preThe obtained data revealed that the nature of the co-contaminant  had a key role in the toxicity mediated by NPls. In recent studies with organic pollutants and MPls in soil it has been shown that the latter can act as adsorbents for hydrophobic organic compounds (HOC), reducing its availability and decreasing the toxicity potential ,54. Howe3) and NMs (VNPs). Effects were observed for both single and combined exposures of VNPs  and AgNO3 (reduction of organisms’ reproduction), in a concentration-related manner. Since these negative effects were similar in single and combined exposures, it showed that no relevant interaction occurred between NPls and VNPs or AgNO3. For these combinations, the presence of NPls did not limit the binary mixture effect. However, considering the combination of NPls with the pharmaceutical, a higher toxicity of NPls + DPH (reproduction decreased) was found compared with the single exposures (no effect). In this case, a significant interaction was observed for NPls and DPH (synergism). The present results showed that the presence of NPls should not be ignored when performing environmental risk assessment of other contaminants. Due to the demonstrated role of NPls as a vector for other contaminants, further studies are encouraged to a more complete understanding about the NPls interactions and mechanisms of toxicity involved when NPls are in complex mixtures of contaminants. One key aspect that should be studied is the observation and characterization of molecular interactions between NPls and other components, such as natural organic matter in complex environmental matrices such as soil, or other pollutant matrices such as wastewater sludge, which require the combination of state-of-the-art techniques. These advances, coupled with the evidence presented in the current paper, will provide important information to fill the knowledge gap on the hazardous potential of NPls to the environment.The present study provided key information on the potential hazard to terrestrial organisms of soil co-contaminated with NPls and other contaminants, specifically pharmaceuticals (DPH), metals (AgNO


We investigated whether family histories of herpes zoster (HZ) are associated with the risk of incident HZ in a Japanese population.A total of 12,522 Japanese residents aged ≥50 years in Shozu County participated in the baseline survey between December 2008 and November 2009 (the participation rate = 72.3%). They were interviewed at baseline by research physicians regarding the registrants’ history of HZ. A self-administered questionnaire survey was conducted to evaluate the potential confounding factors. 10,530 participants without a history of HZ were followed up to ascertain the incidence of HZ during 3-years follow-up until the end of November 2012 with Japanese nationals. We estimated hazard ratios (HRs) of incident HZ according to first-degree family histories using the Cox proportional hazard regression after adjusting for age, sex, and other potential confounding factors.Compared to no HZ history of each family member, a history of brother or sister was associated with a higher risk of incident HZ while histories of father and mother were not. The multivariable HR (95%CI) of incident HZ for a history of brother or sister was 1.67 (1.04–2.69). When comparing to no family histories of all first-degree relatives, the multivariable HRs (95%CIs) were 1.34 (0.77–2.34) for a history of brother or sister alone, but 4.81 (1.78–13.00) for a history of mother plus brother or sister. As for the number of family histories, the multivariable HRs (95%CIs) were 1.08 (0.76–1.54) for one relative  and 2.75 (1.13–6.70) for two or more relatives.Family histories of mother plus brother or sister and two or more first-degree relatives were associated with a higher risk of incident HZ.  Herpes zoster (HZ) is a painful disease caused by varicella-zoster virus (VZV) infection. Patients with HZ usually experience painful vesicular rashes with erythema, which generally take 3–4 weeks to heal . AccordiA family history of HZ has been reported as a risk factor for HZ –10 for wPotential confounding variables other than age and sex were lower body mass index (BMI) , smokingThe goal of the present study was to examine whether first-degree family histories of HZ and the number of these family histories were positively associated with the risk of incident HZ in a free-living general population.A prospective cohort study was undertaken in Shozu County, Kagawa Prefecture, consisting mainly of Shodoshima and Toyoshima Islands with a census population of 33,782, of which 32.8% were 65 years or older on July 1, 2008. The detailed methods of this study have been described elsewhere [The eligible study subjects were 19,058  Japanese residents aged ≥50 years on October 1, 2008. The provisional registration was performed for 12,896 persons by confidential mail, and those who completed the informed consent were formally registered. Eventually, 12,522  were registered between December 2008 and November 2009 .We excluded 1,992  participants who had a past history of HZ, the remaining 10,530  were included in the analysis. The participants’ enrollment process is shown in Figure At baseline survey, an interview was conducted by research physicians to inquire about the participants’ history of HZ and when it had occurred, and whether they had visited a clinic or hospital. A self-administered questionnaire was used to evaluate factors including age, height, weight, and current health status , and family histories of first-degree relatives  of HZ.Telephone surveys were conducted every four weeks to confirm the presence or absence of rash, pain, history of contact with patients with varicella, and admission to a clinic or hospital. Participants with symptoms suspicious for HZ, including rash had been asked to visit a clinic or hospital, and underwent blood tests. We directed most of all participants to consult a physician soon after developing symptoms in the monthly telephone assessments.The duration of the follow-up was between April 2009 and November 2012.Differences in baseline characteristics according to the presence or absence of family histories of HZ were examined using the analysis of variance for mean values and chi-square test for frequency. The crude, age- and sex-adjusted, and multivariable HRs and 95% CIs of incident HZ were calculated according to the family histories using a Cox proportional hazard regression model, adjusting for age, sex, and other confounding variables.The family histories of HZ were categorized into the presence versus absence of each family member, i.e., father, mother, and brother or sister, and the presence of father only, mother only, brother or sister only, father and mother, father and brother or sister, mother and brother or sister, and all of them versus the absence of all relatives. The number of family histories of HZ was grouped into none, one, and two or more of histories for father, mother and brother or sister.2) calculated as the body weight divided by the square of the height, smoking status , drinking status , perceived mental stress , motivation , negative life events (no or yes), and underlying diseases . All statistical analyses were performed using SAS for Windows . P-values for statistical tests were two-tailed, and values <0.05 were considered statistically significant.The confounding factors for the multiple adjustment were BMI  of HZ were 4.50 3.15–6.41) for a history of single blood relative and 13.77 5.85–32.39) for that of multiple blood relatives, compared to no family history [.41 for a5–32.39 f5%CIs of The strength of the present study included a fairly-high participation rate, systematic ascertainment of HZ cases, and the confirmation of HZ diagnosis by polymerase chain reaction detection of VZV . HoweverFamily histories of mother plus brother or sister and two or more first-degree relatives were associated with a higher risk of incident HZ among the free-living Japanese population.


Plasmodium falciparum malaria. One such combination is artemisinin-piperaquine tablets (ATQ). ATQ has outstanding advantages in anti-malarial, such as good efficacy, fewer side effects, easy promotion and application in deprived regions. However, the data about the reproductive and endocrine toxicity of ATQ remains insufficient. Thus, we assessed the potential effects of ATQ and its individual components artemisinin (ART) and piperaquine (PQ) on the reproductive and endocrine systems in Wistar rats.The WHO recommends artemisinin-based combination regimens for uncomplicated The unfertilized female rats were intragastric administrated with ATQ , PQ , ART , or water (control) for 14 days, respectively. The estrous cycle and serum levels of estradiol (E2), follicle-stimulating hormone (FSH), luteinizing hormone (LH), prolactin (PRL), prostaglandin (PG), and adrenocorticotropic hormone (ACTH) were determined. The weights of the kidney, adrenal gland, uterus, and ovaries were measured. The histopathological examinations of the adrenal gland, ovary, uterus, and mammary gland were performed.Compared with the control group, there were no significant differences in the examined items of female rats in the ART groups, including general observation, estrous cycle, hormonal level, organ weight, and histopathological examination. The estrous cycle of female rats was disrupted within 4–7 days after ATQ or PQ administration, and then in a persistent dioestrus phase. At the end of administration, ATQ and PQ at three doses induced decreased PG, increased ACTH, increased adrenal weight and size, and pathological lesions in the adrenal gland and ovary, including vasodilation and hyperemia in the adrenal cortex and medulla as well as hyperplasia and vacuolar degeneration, ovarian corpus luteum surface hyperemia, numerous but small corpus luteum, and disordered follicle development. But the serum levels of E2, FSH, LH, and PRL did not change obviously. These adverse effects in ATQ or PQ treated rats could not completely disappear after 21 days of recovery.Based on the results of this study, ART had no obvious reproductive and endocrine effects on female rats, while ATQ and PQ caused adrenal hyperplasia, increased ACTH, decreased PG, blocked estrus, corpus luteum surface hyperemia, and disrupted follicle development in female rats. These events suggest that ATQ and PQ may interfere with the female reproductive and endocrine systems, potentially reducing fertility. Plasmodium falciparum malaria, is artemisinin-based combination therapy (ACT), which combines artemisinin or its derivatives with a partner drug. Artemisinin and its derivatives are highly potent drugs for multi-drug resistant Plasmodium falciparum treatment. These compounds cause a swift parasite reduction, have broad parasite stage specificity, and are effective against all Plasmodium spp. in humans. Due to the short half-life of artemisinin and its derivatives, combination therapies of an artemisinin-based compound and long-acting antimalarial drugs are gaining importance.Malaria is an infectious disease that seriously endangers human health. It is caused by parasites and spreads to people through the bites of infected female Anopheles mosquitoes. According to the World Health Organization (WHO) world malaria report , there aOne of these combination therapies is the fixed-dose artemisinin-piperaquine tablets (ATQ) initially developed by Chinese scientists. The specification for ATQ is each tablet contains 375 mg PQ and 62.5 mg ART. The clinical usage and dosage of ATQ are as follows: oral administration, the course of treatment is two days, once a day, with an interval of 24 h. 16 years old and above take 2 tablets each time, 11–15 years old take 1.5 tablets each time, and 7–10 years old take 1 tablet each time. ATQ obtained the Chinese New Drug Certificate in 2006 and is protected by patents in 40 countries, including the United States. It has been registered and listed in 22 countries, such as Nigeria and Kenya. In addition, it is listed as the first-line drug for the treatment of malignant malaria by the Chinese Ministry of Health. So far, ATQ has shown good safety and efficacy over a two-day course of treatment [50) of ATQ in Kunming (KM) mice was 2802.38 mg/kg [Previous toxicity studies devoted to investigating acute and sub-acute toxicity of ATQ in rats, dogs, and monkeys. Such as the half lethal dose  was purchased from Tongrentai Pharmaceutical Co., Ltd., Sichuan, China. PQ (Lot No. 130536) and ATQ (Lot No. 20130510) were provided by Artepharm Co., Ltd., Guangdong, China. ATQ, PQ, and ART were ground and then formulated with distilled water to obtain suspensions of different concentrations .Specific pathogen-free grade Wistar female rats aged 7–8 weeks were obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd., China. The animals were allowed to acclimatize for 5 days before the start of the study. Rats were maintained in an environmentally controlled room under standard laboratory conditions of room temperature (22–25 °C) and relative humidity (43–65%) with a 12 h light/dark cycle. The certified commercial feed diet  and drinking water were available ad libitum. All animal experiments were conducted in compliance with the Principles of Good Laboratory Practice (GLP) from the National Medical Products Administration (NMPA), China, and were performed in the laboratory animal room of the New South Center of Safety Evaluation for Drugs of Guangzhou University of Chinese Medicine, China  2018-0014). The research protocol was approved by the Institutional Animal Care and Use Committee (IACUC) for animal care and use based on the 3R principle .The dose design of ATQ was based on the results of a pilot study in female rats. The low, medium, and high doses of ATQ in the pilot rat study were 40, 80, and 160 mg/kg, respectively, of which 160 mg/mg was close to the adult clinical equivalent dose based on the body surface area conversion method. After 14 days of administration, female rats in the three dose groups showed obvious reproductive and endocrine toxicity such as abnormal estrous cycle. Because apparent toxicity has already occurred below the therapeutic dose, and we wanted to find out the dose that does not affect the reproductive and endocrine systems, the doses of ATQ in the current study were adjusted down to 20, 40, and 80 mg/kg. According to the component content of ART and PQ in ATQ, the corresponding three doses were designed to be PQ 15, 30, 60 mg/kg and ART 2.5, 5, 10 mg/kg, respectively.A total of 160 female rats were randomly divided into ten groups with 16 rats per group: control group (water); ATQ low, medium, and high dose groups ; PQ low, medium, and high dose groups ; ART low, medium, and high dose groups . The control group was designed to monitor whether the experimental conditions were normal, and to compare with the treated groups, etc. The separate PQ groups and ART groups were set to distinguish which component of ATQ plays a role when the animals have adverse reactions, the endocrine effect of a single drug on rats, etc.The rats in the ATQ, PQ, and ART groups were orally administered with the corresponding dose of ATQ, PQ, and ART, respectively, once a day for 14 days. Distilled water was given to the rats in the control group. The daily application volume (10 mL/kg body weight) of each individual was adjusted once weekly based on body weight. After 14 days of administration, 10 rats from each group were selected to be sacrificed, the remaining rats were stopping administration and recovered for 21 days. Throughout the study, the rats were observed twice daily for clinical signs, such as appearance, behavior, secretions, excretions, respiration, and other toxicity symptoms. Body weight was measured once a week.After the 5-day acclimation, the estrous cycle of the rats was measured once in the morning and afternoon each day. A wet cotton swab was gently and slowly inserted into the vagina, and the cell sample was removed along the dorsal vaginal wall and then smeared on a glass slide. Papanicolaou was used for staining, and the estrous cycle of the rats was observed under a CX31 biomicroscope  after the smear was dried. The estrous cycle was estimated according to the following criteria. (1) Proestrus: Oval nucleated epithelial cells account for the vast majority, with few white blood cells and keratinized epithelial cells, lasting for 17–21 h. (2) Estrus: Keratinized squamous cells are observed, mostly non-nucleated, while there are cheese-like keratinocytes at the end of estrus, lasting for 9–15 h. (3) Metaestrus: keratinocytes are replaced by small oval nucleated epithelial cells and polymorphonuclear leukocytes, lasting for 14–18 h. (4) Dioestrus: Aged polymorphonuclear leukocytes are seen, along with a small amount of nucleated epithelial cells and mucus, lasting for 48–60 h. The average estrous cycle of female rats is 4–5 days . After tApproximately 0.8 mL of blood was collected from all rats with glass capillary at three time points, two days before administration, at the end of administration, and the end of recovery. After being placed overnight at 4–8 °C, the blood samples were centrifuged at 3000 rpm for 10 min at 4 °C, and the separated sera were stored at -80 °C. Serum levels of follicle-stimulating hormone (FSH), luteinizing hormone (LH), prolactin (PRL), estradiol (E2), adrenocorticotrophic hormone (ACTH), and prostaglandin (PG) were determined by enzyme-linked immunosorbent assay (ELISA). These ELISA kits were purchased from Shanghai Yili Biological Technology Co., Ltd, and used the double antibody sandwich method to specifically detect the corresponding hormone concentrations in rat serum and plasma samples. The sensitivities of the ELISA kits to detect FSH, LH, PRL, E2, ACTH, and PG were 0.7 IU/L, 1.5 pg/mL, 10 pg/mL, 2 pg/mL, 2.5 pg/mL, and 15 pg/mL, respectively. The absorbance values of the samples were measured using an Epoch microplate reader .At the end of administration and recovery, the rats were subjected to 12–16 h fasting, anesthetized with pentobarbital sodium , and euthanized by acute hemorrhage. A complete necropsy was performed on all rats, focusing on the reproductive and endocrine related organs. The weights of the kidney, adrenal gland, uterus, and ovaries were measured. The adrenal gland, uterus, ovaries, and mammary gland were fixed in 10% neutral buffered formalin. These tissues were trimmed, embedded in paraffin, sectioned, stained with hematoxylin and eosin, and then evaluated by an experienced pathologist using a DMLB biomicroscope .P ≤ 0.05.The data were presented as mean and standard deviation (mean ± SD). The statistical analyses were performed using SPSS 19.0 software. If the variance was homogeneous, the difference between groups was evaluated by the one-way analysis of variance (ANOVA) test. If not, the Kruskal-Wallis non-parametric test was used. If either of the tests showed a significant difference between groups, the analysis was continued by the multiple comparison procedure of Dunnett’s test. Differences were considered statistically significant when There was no abnormality in the autonomic activities , sensory function , and neuromuscular function  of the rats in each group. The rats did not have central nervous system toxicity such as convulsion and twitching. Statistical evaluation of the body weights showed no significant difference among groups at all time points during the study.The estrous cycle of all rats before administration was regular and complete, with an average estrous cycle of 4.6 ± 0.8 days. During the administration and recovery period, there was no obvious abnormality in the estrous cycle of rats in the control group and the three dose groups of ART, and each estrous cycle was maintained within 4–5 days. However, the rats in all ATQ and PQ dose groups showed estrous cycle disorder within 4–7 days after administration, and the time in metaestrus was significantly prolonged. Then no estrous manifestation was observed after 7 days of administration, and the estrous cycle maintained in metaestrus to dioestrus. Until the end of the 21-day recovery period, there was still no estrus.Two days before administration, the animals were not yet grouped. The hormonal levels were measured in 160 rats, of which 24, 23, 29, and 84 rats were in proestrus, estrus, metaestrus, and dioestrus, respectively. As shown in Fig. At the end of administration, only 3, 1, 1, and 2 rats were found in estrus in the control group and ART low, medium, and high dose group, respectively, all hormonal values of these rats did not change significantly. Therefore, even though the rats in each group were not all in the same estrous phase, the endocrine hormones were expressed as overall levels.The endocrine hormonal values of female rats in each group at the end of administration are summarized in Table The weights of the kidney, adrenal gland, ovary, and uterus of rats at the end of administration are presented in Table After 14 days of administration, histopathological examination showed no significant changes in the uterus and mammary gland of the treated rats, while the adrenal gland and ovary appeared pathological lesions as illustrated in Fig. The experimental results showed that ART has no significant effect on reproductive and endocrine systems in female rats, since all the examined items were normal, including general observation, estrous cycle, hormonal level, organ weight, and histopathological examination. The female rats in all ATQ and PQ dose groups had no estrus, decreased PG, increased ACTH, increased weights of the kidney and adrenal gland, and pathological lesions of the adrenal gland and ovary. Therefore, most of the reproductive and endocrine effects of ATQ in female rats may come from PQ. The current results of ART were similar to those of a previous reproductive toxicity study in rats , which sIn the current study, the increases in adrenal weight and size of rats in ATQ and PQ groups were mainly due to the hyperplasia of the adrenal cortex and medulla and its vasodilation and hyperemia. Besides, the production of ACTH in the rats of these groups was promoted. Therefore, it is suggested that ATQ and PQ could induce adrenal hyperplasia in rats, which is in correspondence with the results of our previous repeated dose toxicity studies in rats and monkeys. Since adrenal hyperplasia can lead to blood electrolyte disorder, the serum biochemical results showed that potassium and chloride decreased significantly after oral administration of ATQ at doses of 63, 126, and 252 mg/kg in rats for 28 days (unpublished data), while a significant decrease in serum sodium and hypertrophy of adrenal cortical parenchymal cells were observed after oral administration of ATQ at doses of 39.1, 78.2, and 156.4 mg/kg in monkeys for 21 days [Adipose organs are mainly composed of white adipose tissue and brown adipose tissue. The color distinction between a “brown” and a “white” adipocyte largely reflects the many more mitochondria (which are high in iron) in brown adipocytes compared to white adipocytes. White adipose tissue stores energy in the form of triglycerides and secretes hormones and cytokines that affect energy balance, while brown adipose tissue consumes energy through the coupling of lipid oxygen in mitochondria to produce heat. The function of brown fat to produce heat is the result of the specific expression of uncoupling protein 1 (UCP1). The brown fat exists within small mammals in distinct locations being innervated by the sympathetic nervous system (SNS). During cold stress, brown fat thermogenesis is classically stimulated by norepinephrine released from SNS, which activates β3-adrenergic receptors in brown adipocytes –23. SealPrevious studies have shown that quinolines cause estrous cycle disorders and ovulation abnormalities in animals. Oral administration of chloroquine for four weeks was found to alter the estrous cycle in rats (i.e. the rats showed a persistent dioestrus smear), lower serum estrogen and LH levels, while serum FSH was unaltered . Gavage Unfortunately, progesterone was not detected in this study, which is important in determining the functional status of the corpus luteum. Pathological examination results showed that the corpus luteum of female rats in ATQ and PQ groups were congested and smaller, suggesting the function of corpus luteum might be impaired, leading to the change of progesterone. Thus, progesterone is recommended to be included in monitoring in the subsequent clinical application of ATQ.Before the study, we supposed that after 14 days of dosing, rats might experience some responses similar to or related to galactorrhea of young nulliparous. However, there was no lactation, pathological damage to the mammary gland, or increased PRL in the treated female rats. Of note, the clinical incidence of ATQ-induced galactorrhea in girls is low (0.04%), the number of animals in our experiment is small, and there are species differences between rats and humans. So these may be the reasons for the absence of lactation-related endocrine changes in the treated rats. In addition, the serum levels of E2, FSH, and LH were also unaltered, but it cannot be excluded that these hormones are not affected after the drug treatment in humans. Therefore, when ATQ or PQ-containing drugs are used in special populations or reproductive endocrine-related adverse reactions occur after treatment , it is recommended to monitor these hormones to fully understand the endocrine status in patients.According to the results presented in this study, ART had no obvious reproductive and endocrine effects on female rats, while ATQ and PQ caused adrenal hyperplasia, increased ACTH, decreased PG, blocked estrus, corpus luteum surface hyperemia, and disrupted follicle development in female rats. These events suggest that ATQ and PQ may interfere with the female reproductive and endocrine systems, potentially reducing fertility.


A 48-year-old Mongolian man developed bilateral leg edema after suffering from a fever for three months. He lost his appetite, and the edema gradually spread from the legs, becoming systemic. In addition, he had difficulty in moving. He had a history of being diagnosed with numerous venous malformations and Kasabach-Merritt syndrome when he was a child. On arrival, he had numerous venous malformations over pale skin, edema at each extremity, and anemic conjunctiva. Chest roentgen showed bilateral pleural effusion, and cardiac echography findings showed a left ventricular ejection fraction of 30% with diffuse hypokinesis. The results of a blood analysis showed coagulopathy, which was compatible with disseminated intravascular coagulation and pancytopenia. He was diagnosed with blue rubber bleb nevus syndrome with Kasabach-Merritt syndrome and heart failure. Use of diuretics, thiamine, iron, phytonadione, carbazochrome, and tranexamic acid, in addition to intermittent transfusion resulted in the improvement of his Kasabach-Merritt syndrome. Radical management of blue rubber bleb nevus syndrome was deemed impossible by dermatologists due to the large amount of venous malformations. We encountered an extremely rare case of blue rubber bleb nevus syndrome with Kasabach-Merritt and heart failure. Multimodal therapy might help manage Kasabach-Merritt syndrome following improvement in coagulopathy and pancytopenia. TEK gene) in many patients with blue rubber bleb nevus syndrome [Blue rubber bleb nevus syndrome is a rare condition, with an estimated prevalence of 1 in 14,000 births . Soblet syndrome .Kasabach-Merritt syndrome is a rare but life-threatening coagulopathy of infancy that presents with thrombocytopenia, microangiopathic hemolytic anemia, and consumptive coagulopathy in the setting of a rapidly enlarging vascular tumor or malformation . The mecWe herein report a case of blue rubber bleb nevus syndrome with Kasabach-Merritt syndrome and heart failure.A 48-year-old Mongolian man developed bilateral leg edema after suffering from a fever for three months. He lost his appetite, and the edema gradually spread from the legs to a systemic state. In addition, he had difficulty in moving, and therefore he was transported to a local hospital by family car.The patient presented to our hospital on the same day because he had heart failure with numerous venous malformations in the skin and DIC. He had a history of having been diagnosed with numerous venous malformations and Kasabach-Merritt syndrome when he was a child. However, he had not undergone a regular medical checkup because he had a fear of hospitals. He was unemployed and did not smoke or drink alcohol. His father had hyperlipidemia and pulmonary disease, his mother had essential thrombocythemia and arrhythmia, and his brother was healthy.On arrival, he had clear consciousness, and his blood pressure was 94/68 mmHg, heart rate was 92 beats per minute, respiratory rate was 20 breaths per minute, and body temperature was 37.4°C. A physical examination revealed numerous venous malformations over pale skin Figure , edema aChest roentgen showed bilateral pleural effusion, an electrocardiogram showed low voltage, and cardiac echography findings showed a left ventricular ejection fraction to 30% with diffuse hypokinesis. The results of blood analysis are shown in Table Whole-body computed tomography (CT) showed a mediastinal cyst, bilateral atelectasis with pleural effusion, ascites, and multiple calcifications in the subcutaneous tissue. He received a diagnosis of blue rubber bleb nevus syndrome with coagulopathy and pancytopenia due to Kasabach-Merritt syndrome and heart failure of unknown etiology.The main time course of the blood tests and treatments is shown in Figure Initially, he received furosemide, spironolactone, and thiamine for heart failure, and transfusion of 4 units of red blood cells and 6 units of fresh frozen plasma for coagulopathy and severe anemia on day 1. The diuretics worked well, and his coagulopathy improved to a degree sufficient for transfusion. However, transfusion of red blood cells did not induce an increase in the hemoglobin level as we expected. He then underwent transfusion of 2 units of red blood cells on day 2, but this was ineffective. As he had microcytic anemia and a low level of iron  μg/dL) and ferritin  ng/mL), he underwent administration of iron and azosemide from day 4. As he showed temporary deterioration of anemia and persistent mild coagulopathy, the administration of phytonadione from day 8 and carbazochrome and tranexamic acid from day 9 were started. He had a low level of vitamin B1 on arrival . On day 8, esophagogastroduodenoscopy did not show the bleeding source, but multiple venous malformations in the esophagus and stomach were found  showed vein of Galen aneurysmal malformation Figure .Systemic edema and dyspnea at rest improved gradually, but dyspnea on rehabilitation remained; therefore, the patient underwent transfusion of 2 units of red blood cells on days 10 and 15, respectively. After these transfusions, his hemoglobin level reached 6 g/dL. Colonoscopy on day 16 did not show the bleeding source but did reveal multiple venous malformations in the colon Figure . On day On day 36, he was able to climb a step ladder. After his hemoglobin level improved to 8g/dL, he was discharged on foot on day 44. The medications prescribed at discharge were 150 mg of iron citrate, 30 mg of carbazochrome, 750 mg of tranexamic acid, 2.5 mg of enalapril, 30 mg of azosemide, 25 mg of spironolactone, and 10 mg of esomeprazole per day. Radical management of blue rubber bleb nevus syndrome was deemed impossible by dermatologists due to the large amount of venous malformations.At one month after discharge, echography and blood tests showed further improvement of his left ventricular ejection fraction to 55%, white blood cells to 3,200/mm3, hemoglobin to 9.7 g/dL, platelets to 16.7 × 104/mm3, prothrombin time to 12.5  seconds, activated partial thrombin time to 32.7  seconds, fibrinogen to 90 mg/dL, fibrin degradation products to 92 μg/mL, iron to 201 μg/dL, and ferritin to 45 ng/mL.The present case was one of blue rubber bleb nevus syndrome with Kasabach-Merritt and heart failure. To our knowledge, there have been two case reports on the combination of blue rubber bleb nevus syndrome and Kasabach-Merritt syndrome, making the present case the third to be described ,6. All tOne of the characteristics of the present case was that the coagulopathy showed an improving trend following the transfusion of fresh frozen plasma; however, the anemia showed no such trend following transfusion of red blood cells. In the present case, iron for iron deficiency anemia, diuretics  and thiamine for heart failure, phytonadione for the prolonged prothrombin time, tranexamic acid for the increased fibrinolysis, and carbazochrome for the vascular hyperpermeability were administered. Tranexamic acid stabilized the coagulative and fibrinolytic parameters and relieved the bleeding tendency in a patient suffering from aortic dissection with an acute exacerbation of chronic DIC . In addiConcerning the heart failure in the present case, there is one previous report describing blue rubber bleb nevus syndrome complicated by heart failure of unknown cause in an elderly Japanese woman . At presWe encountered an extremely rare case of the combination of blue rubber bleb nevus syndrome and Kasabach-Merritt and heart failure. There have been two case reports concerning the combination of blue rubber bleb nevus syndrome and Kasabach-Merritt syndrome, making the present case the third to be described. All three cases were Japanese, and therefore a genetic factor might be involved in this combination.The coagulopathy showed an improving trend following the transfusion of fresh frozen plasma; however, the anemia showed no such trend following transfusion of red blood cells.Multimodal therapy might contribute to the improvement of Kasabach-Merritt syndrome following improvement of coagulopathy and pancytopenia.


Background: Lafora disease (LD) is a neurodegenerative condition characterized by the accumulation of polyglucosan bodies (PBs) throughout the brain. Alongside metabolic and molecular alterations, neuroinflammation has emerged as another key histopathological feature of LD. Methods: To investigate the role of astrocytes and microglia in LD, we performed a systematic review according to the PRISMA statement. PubMed, Scopus, and Web-of-Science database searches were performed independently by two reviewers. Results: Thirty-five studies analyzing the relationship of astrocytes and microglia with LD and/or the effects of anti-inflammatory treatments in LD animal models were identified and included in the review. Although LD has long been dominated by a neuronocentric view, a growing body of evidence suggests a role of glial cells in the disease, starting with the finding that these cells accumulate PBs. We discuss the potential meaning of glial PB accumulations, the likely factors activating glial cells, and the possible contribution of glial cells to LD neurodegeneration and epilepsy. Conclusions: Given the evidence for the role of neuroinflammation in LD, future studies should consider glial cells as a potential therapeutic target for modifying/delaying LD progression; however, it should be kept in mind that these cells can potentially assume multiple reactive phenotypes, which could influence the therapeutic response. EPM2A [EPM2B genes [Lafora disease (LD) is a severe neurodegenerative condition belonging to the group of progressive myoclonic epilepsies (PME). It is a rare genetic disorder linked to mutations in the EPM2A  and EPM22B genes . LD’s hi2B genes ,5. Despi2B genes ,7 that m2B genes ,7. These2B genes ,12,13,142B genes ,16,17, t2B genes , increas2B genes ,20.In the recent years, neuroinflammation has also emerged as a key histopathological feature in animal models of LD ,21,22,23In this review we systematically examined studies investigating the roles of glial cells in neuroinflammation in LD. Several studies have claimed a contribution of astrocytes and microglia in LD, starting with the evidence that these cells also accumulate PBs ,39,40,41https://www.zotero.org/download/, accessed on 12 October 2022) to collect all the results in a single library. Abstracts were retrieved using our search strategy, and duplicates were removed. The full text of all potentially eligible articles and their A systematic literature review was conducted using the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines . The PubThe PRISMA flow chart of the review process is presented in postmortem histopathological studies on the brains of individuals [Epm2b−/−), Valles-Ortega and colleagues [Epm2a−/− and Epm2b−/− mice confirmed that PBs colocalize in neurons, astrocytes, and microglial cells [The PBs found in LD have long been assumed to accumulate only in neurons , which sividuals ,48 and dividuals ,57 have lleagues  showed tlleagues . A subseal cells  and thatal cells . Interesal cells . Researcal cells . These tal cells . In partal cells , which sal cells . The proal cells . Furtheral cells .Alongside the accumulation of PBs in both neuronal and glial cells, neuroinflammation has emerged as another key histopathological feature in LD animal models ,22. It aEpm2a−/− and Epm2b−/− mice at 16 months of age [Our analysis of data from transcriptomic  and PCR s of age . Anothers of age .The presence of astrogliosis and microgliosis was also confirmed by histopathological studies on brain sections from dogs with LD ,50.gfap also confirmed astrocyte involvement in −/−epm2a zebrafish larvae [tnfa, cox2b, and il1b and genes such as csfr1a suggests the presence of proinflammatory activated cells [csfr1a, in particular, this upregulation may, through a positive feedback mechanism, help to support the DAM signature by interacting with trem2 signaling [The early activation of glial cells has also been observed in a laforin-deficient zebrafish model , while th larvae . Furthered cells ,77; in tignaling . Finallyignaling .The question of what triggers the activation of glial cells in LD is still debated. It is very likely that multiple factors  play a part, especially considering that these are very active cells that are capable of responding to minimal changes in their environment ,80.The inhibition of glycogen synthesis has been shown to have positive effects on neuroinflammation, suggesting a possible role of PB accumulation in glial cell activation. Positive effects on neuroinflammation were also obtained using drugs with anti-inflammatory actions or acting on other molecular/metabolic pathways found to be impaired in LD ,23,62. TInteresting results emerged from the larval zebrafish model of LD . Up to 5Albeit variable in degree, LD mice exhibit neurodegeneration with neuronal loss and the activation of astrocytes and microglia ,41,46,83+ and glutamate released during neuronal activity, which they perform through the Kir4.1 channel and the EAAT2 transporter [One of the most important homeostatic roles of astrocytes is the clearance of the Knsporter ,91. If tnsporter ,93. Indensporter ,69,94,95nsporter ,97,98. Ansporter  and mousnsporter  of LD isnsporter ,53 linkensporter .Another important glial cell function is the trophic support provided to neurons through the release of several growth factors  ,88,99. PAlongside this loss of their homeostatic functions, glial cells activated in a proinflammatory sense may acquire neurotoxic properties . ProinflProinflammatory cytokines  released by both proinflammatory microglia and astrocytes  can causThe observation of the upregulation of the complement system, in particular of the components C1q  and C3 fCOX-2, increased in several models of LD, contributes to neuroinflammation and possibly neurodegeneration through the production of prostanoids . AlthougIt is also important to remember that immune cells, including microglia, play an important role in the regulation and distribution of brain iron. In addition, inflammatory cytokines such as TNF-α and IL-6 induce the expression of iron transport receptors and promote iron accumulation in neurons and microglia, which may contribute to neuroinflammation . TherefoData from the studies of the mouse and zebrafish models of LD included in this review suggest that a portion of glial cells may exhibit an anti-inflammatory phenotype that, contrary to what has been seen so far, could have a neuroprotective effect. To date, however, this hypothesis has not been fully investigated.The mechanisms underlying LD epilepsy remain unknown. Since, over time, several authors have argued that it may have a multifactorial origin, in this review we focus on the possible contribution of glial cells to epileptogenesis in LD.In animal models of LD, all drugs ,47,61,62Overall, the literature seems to support a role for neuroinflammation in LD epilepsy, be it driven by PBs or other mechanisms . Neuroinflammation and epilepsy are known to influence each other ,126. Dat+ and glutamate clearance, as seen above, can lead to neuronal hyperexcitability and contribute to epileptogenesis. In this regard, in addition to the aforementioned data on EAAT2 [The mechanisms by which glial cells may contribute to LD epilepsy are multiple. First, impaired Kon EAAT2 ,53,54, won EAAT2 . The dowon EAAT2 , and we on EAAT2 . Many ofon EAAT2 ,129. Indon EAAT2 ,105 and on EAAT2 , which rThis paper systematically reviews published data dealing specifically with the role of astrocytes and microglia in neuroinflammation in LD. Although LD has long been approached from a “neuronocentric” perspective, there is growing evidence suggesting that glial cells also play a part in its disease mechanisms, in line with what is seen in most neurological ,130 and The first important observation emerging from this data review concerns the discovery of PBs in glial cells ,40,41,51The drivers of the proinflammatory polarization of glial cells are only partially known and are potentially numerous. Whereas the accumulation of PBs in astrocytes has been shown to activate both astrocytes and microglia in a proinflammatory manner, pharmacological studies in LD mouse models have suggested that neuroinflammation may also depend on other pathways, such as impaired autophagy , increashexb gene upregulation in a zebrafish model of LD [In short, the role of microglia in LD is still underexplored, and inferences regarding this type of cell in LD are limited by the lack of targeted studies. Although PBs have also been observed in this cell type, we do not know whether they accumulate glycogen autonomously or whether they phagocytize it from outside. Certainly, characterizing microglial glycogen inclusions  could help to clarify some issues. The finding of mediators and markers of anti-inflammatory pathways in mouse LD models ,39 suggeel of LD  suggestsel of LD ,11,12,13el of LD ,63,66 anAlthough the recent literature is revealing knowledge gaps, and many questions remain unanswered, glial cells are clearly involved in LD. Future studies should be designed to address glial cells as a potential therapeutic target, with the aim to arrest the progression of LD. In this context, the variability in the polarization of astrocytes and microglia observed within the same disease could make treatment with anti-inflammatory drugs difficult and not entirely effective. For this reason, it is important to characterize the phenotypes of these cells in more detail and possibly combine strategies aimed at enhancing the anti-inflammatory/protective processes. Although animal models remain the ideal platforms to continue studying the disease and for drug screening, it would also be useful to study microglial and astrocytic activation in humans through fluid ,138,139