Periodontitis is a chronic immuno-inflammatory disease characterized by inflammatory destruction of tooth-supporting tissues. Its pathogenesis involves a dysregulated local host immune response that is ineffective in combating microbial challenges. An integrated investigation of genes involved in mediating immune response suppression in periodontitis, based on multiple studies, can reveal genes pivotal to periodontitis pathogenesis. Here, we aimed to apply a deep learning (DL)-based autoencoder (AE) for predicting immunosuppression genes involved in periodontitis by integrating multiples omics datasets.Two periodontitis-related GEO transcriptomic datasets (GSE16134 and GSE10334) and immunosuppression genes identified from DisGeNET and HisgAtlas were included. Immunosuppression genes related to periodontitis in GSE16134 were used as input to build an AE, to identify the top disease-representative immunosuppression gene features. Using K-means clustering and ANOVA, immune subtype labels were assigned to disease samples and a support vector machine (SVM) classifier was constructed. This classifier was applied to a validation set (Immunosuppression genes related to periodontitis in GSE10334) for predicting sample labels, evaluating the accuracy of the AE. In addition, differentially expressed genes (DEGs), signaling pathways, and transcription factors (TFs) involved in immunosuppression and periodontitis were determined with an array of bioinformatics analysis. Shared DEGs common to DEGs differentiating periodontitis from controls and those differentiating the immune subtypes were considered as the key immunosuppression genes in periodontitis.We produced representative molecular features and identified two immune subtypes in periodontitis using an AE. Two subtypes were also predicted in the validation set with the SVM classifier. Three “master” immunosuppression genes, PECAM1, FCGR3A, and FOS were identified as candidates pivotal to immunosuppressive mechanisms in periodontitis. Six transcription factors, NFKB1, FOS, JUN, HIF1A, STAT5B, and STAT4, were identified as central to the TFs-DEGs interaction network. The two immune subtypes were distinct in terms of their regulating pathways.This study applied a DL-based AE for the first time to identify immune subtypes of periodontitis and pivotal immunosuppression genes that discriminated periodontitis from the healthy. Key signaling pathways and TF-target DEGs that putatively mediate immune suppression in periodontitis were identified. PECAM1, FCGR3A, and FOS emerged as high-value biomarkers and candidate therapeutic targets for periodontitis. Periodontitis involves the inflammatory destruction of the supporting tissues of teeth. It involves a perturbed local host immune response that is ineffective in countering plaque biofilm microbiota . Innate Experimental studies are limited by scale, incomplete or inaccurate existing databases, and the cost-intensive nature of molecular experiments, so approaches that can predict previously unidentified gene functions, enable gene function discovery, and automate the identification of inaccuracies can be very valuable . Deep leTo our knowledge, the present study is the first to integrate multi-omics data pertaining to immunosuppression genes in periodontitis using a DL-based AE combined with a support vector machine (SVM) classifier  confirmeF values. Data partitioning of the inferring samples of GSE16134 was applied to assess the robustness of the AE, using a 5-fold CV. The samples were randomly split into 5 folds, 3 of which were used as the training set (60%) and the remaining 2 (40%) as the test set. Thereafter, based on the clustering results and the top 100 genes of GSE16134, a SVM classifier was built with a 5-fold CV to identify the optimal hyperparameters, and a validation set (immunosuppression genes related to periodontitis in GSE10334) was applied for SVM to predict the subtypes. To explore the biological roles of the different identified subtypes, differentially expressed genes (DEGs) and transcription factors (TFs), differential expression analysis, functional enrichment analysis, and construction of TF-target DEGs interaction network were, respectively, applied. Finally, to identify the immunosuppression genes that might be most pertinent to periodontitis, the overlapping DEGs among the DEGs discriminating periodontitis and controls and DEGs discriminating the subtypes classified with the DL-based model were determined.An overview of the workflow of this study is depicted in 1. Detailed information of the two datasets is listed in 2 and HisgAtlas3. From these obtained genes, 1,207 immunosuppression genes related to periodontitis were extracted. Next, the two datasets were stacked, and 1,181 immunosuppression genes’ expression profiles were found matching in the two datasets. Subsequently, the two datasets were standardized using the “scale” function in R, setting the parameters as .Transcriptomic data from gingival tissue samples affected with periodontitis and the corresponding controls (GSE16134 and GSE10334) were obtained from the Gene Expression Omnibus (GEO) database of NCBI4”. An AE is a three-layered neural network consisting of input, hidden, and output layers  were set to 0.00002 and 0.00001. In addition, the AE was trained using the gradient descent algorithm with 20 epochs and 50% dropout. Here, an epoch is an iteration that indicates the number of passes of the entire training dataset, while the size 20 is one of the appropriate training cycles calculated in the evaluation of the model.The 100 nodes from the bottleneck-hidden layer were considered as new features for the analysis and were clustered with the K-means algorithm. The optimal number of clusters was determined based on two metrics: Silhouette index  and CaliPrincipal component analysis (PCA), a conventional dimension reduction approach was applied to compare with the AE performance . The samData partitioning of the inferring samples of GSE16134 was done to assess the robustness of the model, using a cross-validation (CV)–like procedure, as described in earlier reports . First, F values. Since the top 100 genes were also present in GSE10334 dataset, a complementation test for missing genes was not conducted. Subsequently, based on the labels assigned using GSE16134, a SVM classifier was built and further applied for prediction in a validation set (GSE10334). The “scikit-learn” package . Then, the top 100 “most relevant” immunosuppression genes in GSE16134 were selected based on the clustering labels and analysis of variance (ANOVA)  package  was used5. The AUC is the area under the ROC curve, where an AUC value above 70% is considered acceptable (Accuracy and area under the curve (AUC) were selected as two metrics to evaluate the performance of the SVM classifier. The percentage of accuracy was calculated as: Accuracy (%) = Predict number / Test number. A receiver operating characteristic (ROC) curve was plotted for the model using the “pROC”  and the ceptable .P value < 0.05, and |log FC| ≥ 1 was selected as differentially expressed genes (DEGs). The DEGs with Log FC ≥ 1 was defined as up-regulated DEGs, while the DEGs with log FC ≤ −1 were defined as down-regulated DEGs.Differential expression analysis was performed for each of the datasets (GSE16134 and GSE10334), to identify genes discriminating between the disease and control samples, using the “Linear Models for Microarray data” (“limma”) package in R . Genes wP value < 0.05, and |log FC| ≥ 0.05 were selected as DEGs; The DEGs with Log FC ≤ 0.05 were defined as up-regulated DEGs, while the DEGs with log FC ≤ −0.05 were defined as down-regulated DEGs.Differential expression analysis was also similarly conducted for the classified subtypes. Here, genes with To identify the most critical immunosuppression genes in periodontitis, the DEGs discriminating disease and control samples that overlapped with DEGs discriminating the different subtypes were identified and visualized using a Venn diagram. To evaluate the performance of each such identified gene, a ROC curve was plotted as described earlier.P value < 0.05 were regarded as significant functions. The top 30 of the enriched GO/BPs and pathways were chosen to be visualized in a bar plot.The DEGs overlapping in the two datasets (GSE16134 and GSE10334) were identified using the “ClusterProfiler” package in R . The funP value < 0.05 were regarded as significant functions. The top 20 of the enriched pathways were listed and visualized using the heatmap function in R  were extracted and used for constructing the TFs-target DEGs interaction network. The network was visualized using Cytoscape (Version 3.7.2)  , and thek = 2, the average silhouette width was nearest to 1  was the highest  were split at a 60%/40% ratio for the training set and testing set. The SVM model presented an accuracy of 92.78% , and theDifferential expression analysis was applied to the disease and control samples, as well as the two classified subtypes. A total of 236 DEGs consisting of 48 down-regulated DEGs and 188 up-regulated DEGs were identified from the GSE16134 dataset, while a total of 194 DEGs consisting of 42 down-regulated DEGs and 152 up-regulated DEGs were identified from the GSE10334 dataset . For disSignificantly enriched biological processes and signaling pathways related to the immunosuppressive DEGs were identified from those overlapping between GSE16134 and GSE10334. The immunosuppressive DEGs involved in periodontitis were implicated in biological processes, including T cell activation, regulation of lymphocyte activation, regulation of T cell activation, regulation of cell-cell adhesion, and leukocyte cell-cell adhesion . The immE. coli bacteria  gene expressed in various cells, such as endothelial cells (ECs), platelets, and immune cells. PECAM1 is found to be a co-modulator of T-cell immunity  and a prf PECAM1 . A protef PECAM1 . FCGR3A f PECAM1 . Previouf PECAM1 . Besidesf PECAM1 . FOS wasOf the top 10 hub TFs, six “leader” immunosuppressive TF-target DEGs with plausible literature evidence were identified as key to periodontitis pathogenesis and included the down-regulated TFs , as well as up-regulated TFs . NFKB1, also termed NF-κB, is a core TF implicated in immune and inflammatory diseases . PeriodoE. coli bacteria, targeting the down-regulated TFs, such as NFKB1, FOS, and JUN  -STAT, and HIF1, are found to activate up-regulated TFs, such as HIF1A, STAT4, and STAT5B . Whereas and JUN . Immune P. gingivalis can lead to the up-regulation of PDL1 expressed by gingival keratinocytes, as well as the overexpression of PD1 expressed on T lymphocytes (Considering PDL1/PD1 signaling that characterized the subtype S1, it has been found that peptidoglycans from phocytes . The intphocytes . As PDL1phocytes , it may phocytes . HIF1A pphocytes , and tumphocytes . Manipulphocytes . The MAPphocytes . Furtherphocytes . Of notephocytes  and Tumophocytes , indicatAltogether, using the DL-based predictive model and bioinformatic analysis, our study provides a predictive and theoretical description of functions and mechanisms relevant to immunosuppression genes active in periodontitis pathogenesis. The validated efficiency and accuracy of the DL-model overcome the bottlenecks of current evidence and suggest new insights valuable for potential translation in therapeutic gene targeting. However, considering our study is the first to apply DL methods in the periodontal disease context, it is expected that further well-designed investigations can validate the model considering other aspects of periodontal disease, where specific and precise associations between clinical parameters and target genes might be identified. One caveat of our study is the lack of phenotype information about the periodontitis cases which were grouped into two distinct immune subtypes. Periodontitis is well recognized as a multifactorial disease, where a disease phenotype may result from multiple factors in a “sufficient cause model” . DistincThe DL-based model applied in this study was reliable and robust in predicting immunosuppression genes in periodontitis. An array of pathways and TF-target DEGs were found to be implicated in the immunosuppressive activity during periodontitis. Three “master” immunosuppression genes, PECAM1, FCGR3A, and FOS, were identified as critical to immune suppression occurring during periodontal pathology. Taken together, the DL model revealed novel insights into the molecular mechanisms underpinning periodontitis and identified key candidate genes for further translation in the context of risk profiling and therapeutic development.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding authors.WN conceived of the presented idea, designed the overall study workflow, analyzed the data, prepared the figures and tables, authored and reviewed drafts of the manuscript, and approved the final draft. AA prepared the figures and tables, was involved in the proofreading and deep editing, and approved the final draft. ZS analyzed the data, prepared the figures and tables, and was involved in the discussion of the results. AO analyzed the data, prepared the figures and tables, and was involved in proofreading and deep editing. CL, SH, QO, and MZ were involved in the discussion of the results, and also prepared the figures and tables. XL and YD designed the overall study workflow, analyzed the data, and prepared the figures and tables. RH, DZ, and GS devised the main conceptual idea, supervised the whole work, and approved the final draft. GP, YW, and XH supervised the whole project, and approved the final draft. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Haplocladium angustifolium was recognized as a good biomonitor of atmospheric deposition in central China by investigating bryophyte communities and habitat environment in various ecological function regions of the urban areas in Wuhan. The concentrations of trace elements, including As, Cd, Co, Cr, Cu, Mn, Ni, V, Pb, and Zn, in moss and soil samples from 25 sampling sites were analyzed by inductively coupled plasma mass spectrometry. The concentrations of Cd and Zn in Haplocladium angustifolium collected from the entire study area were much higher than those in substrate soil. Cd was at the highest ecological risk level among the 10 elements, which contributed 34.5% to the potential ecological risk index (RI). An RI value of 392.8 indicated that urban atmospheric quality in Wuhan was in a considerable potential ecological risk. The index of atmospheric purity, regarding species richness, cover, and frequency of bryophytes, was spatially and negatively correlated with RI, also demonstrated the atmospheric quality. Effective measures should be considered to alleviate certain airborne trace element contamination and protect the environment and human health in this metropolis.Bryophytes are popular biomonitoring plants for atmospheric environments. The objectives of this study were to examine the characteristics of bryophyte communities, determine a suitable monitor species, and assess urban atmospheric environment quality by the joint use of bryophyte features and chemical properties in a large city in China. A pleurocarpous feather moss  Atmospheric pollution due to population growth and shift, the increases in urbanization and industrialization, and the continuous increase of motorized transportation is one of the major problems in urban areas and is a source of great public concern regarding environmental and health consequences ,2,3. VarHypnum cupressiforme, Haplocladium microphyllum, Pleurozium schreberi, and Pseudocleropodium purum [When using naturally growing bryophytes as biomonitors, either the composition and structure of bryophyte communities  and the development of certain bryophytes is considered , or certum purum ,15,16. Tum purum ,18. The um purum ,20.Biomonitoring techniques by bryophytes have been widely employed worldwide, especially in Europe ,22. In CThe study was conducted in the urban areas of Wuhan City, Hubei Province, China. Wuhan is a metropolis located at latitude 29° 58′–31° 22′ N and longitude 113° 41′–115° 05′ E. It is recognized as the main political, economic, financial, cultural, educational, and major transportation hub in central China, with a large population of 11.08 million. Abundant water bodies are distributed in the city and cover 26.1% of the city areas. Elevations of most city areas are below 50 m. The climate is characterized as subtropical, with distinct seasonal divisions, annual mean air temperature of 15.9 °C, and annual precipitation from 1100 mm to 1300 mm. As a major transportation hub, dozens of railways, roads, and expressways pass through Wuhan. Heavy industries, including steel works, chemical plants, and power plants, are also under development. Thus, the urban area of this city has mainly faced pollution from transportation and industry in recent decades.Thirteen administrative divisions are included in Wuhan, of which seven divisions  are considered urban areas . Twenty-Flora Bryophytarum Sinicorum Vol.2–8 [Flora Yunnanica Vol. 17 [Bryophyte species were identified according to  Vol.2–8 ,31,32,33 Vol. 17  in the lHaplocladium angustifolium, a dominant species with widespread distribution and high abundance) and soil samples were prepared for trace element analyses. Bryophyte samples (dominant species with widespread distribution and high abundance) were prepared by manually removing soil particles, dead materials, and litters. The green or greenish-brown parts of the bryophytes from dust particles were cleaned with deionized water. The bryophyte and soil samples were dried to a constant weight in a thermostatic drying machine for 48 h at 40 °C. The bryophytes were then ground to acquire fine powder in a mill, and the soil samples were homogenized with a mortar and pestle after coarse materials were removed using a 2 mm sieve. Three replicate measurements per bryophyte and soil sample were kept in clean, dry paper bags for further analyses.Moss samples (3:H2O2 = 4:1), and 0.25 g of each soil sample was digested with 10 mL of mixed acid (HNO3:HCl:HF = 3:1:1) for 30 min and then moved to a microwave oven  for enhanced digestion until transparent solutions were obtained. After cooling, the digests were transferred to a 50 mL volumetric flask. The bryophyte was then filled with deionized water to 25 mL and soil to 50 mL. The presence and concentrations of trace elements were determined by inductively coupled plasma mass spectrometry . The concentration of each element was corrected by subtracting blank values. A blank and a plant standard GBW07603  or a soil standard GBW07403  were analyzed to check the accuracy and precision of each element analysis. The recovery percentages of elements were >85% for quantitative analysis. All the detailed procedures of samples preparation and chemical analyses were referred to our previous study [Approximately 0.5 g of each bryophyte sample was transferred into a digestion tube and cold digested with 10 mL of mixed acid  relationships were characterized by canonical correspondence analysis (CCA). Suitable biomonitoring species for tracing atmospheric trace elements were identified in this study on the basis of having high cover, frequency and importance value, and being widely distributed as recognized by CCA. CCA and the corresponding 2-dimensional ordination graphs were implemented in software CANOCO for Windows 4.5 .The IAP  based on and Cao .The potential ecological risk index (RI), which reflects the potential ecological harm from a single metal and considers the integrated ecological effect of multiple elements ,38, is aectively . The conly [Cni) . The clat test. The intercorrelations among site-specific elements, IAP and RI, were characterized by Pearson’s correlation analysis. The Shapiro–Wilk test of normality was conducted before parametric statistical analyses; the non-normal distribution dataset was transformed by log transformation.The concentration values of trace elements from bryophyte and soil samples were given as minimum, maximum, mean, standard deviation (SD), and coefficient of variation (CV) for the 25 sampling sites. The statistical differences of each element concentration between the bryophyte and soil samples were determined by a paired-samples At 25 sampling sites, 90 bryophyte species in 34 genera from 19 families were surveyed, with 67 species found on the ground and 44 species noted on the trunk of 61 trees. The families with the highest number of species present were Pottiaceae and Bryaceae, with almost one third of all surveyed species richness . The speWithin the 25 sampling sites, no epiphytic bryophytes species were found in 11 sites, such as N2, N4, N9, and N10. By contrast, N19, N24, and N25 sites showed a larger number of ground and epiphytic bryophyte species than the other sites a. Eight H. angustifolium, which is a member of Thuidiaceae. This species has creeping main stems and pinnate branching systems, and the coverage and frequency were much higher than those of other species in the urban areas of Wuhan (H. angustifolium (S1) seemed to have no preference and was distributed in all sampling sites, on the ground, and tree trunk . The mean values of the trace element concentrations were in the following order: Mn > Zn > V > Cu > Cr > Pb > Ni > Co > As > Cd, with the Mn and Zn concentrations higher than 200 mg kg−1 and Cd levels lower than 1 mg kg−1. Although the concentrations of As and Cd were much lower than Mn and Zn, their contamination coefficients were much higher ; thus, they may be in a higher risk level. Comparatively, the concentrations of Cd and Zn in moss samples were significantly higher than those in soil samples (p < 0.01), whereas those of As, Cr, Mn, Pb, Ni, and V were significantly lower (p < 0.01). Among the 10 elements, the concentrations of As, Cd, Cu, Pb, and V in moss were significantly correlated with those in the substrate soil .Trace elements in the samples of H. angustifolium are presented in (p < 0.01). Cd was significantly correlated with Pb and Zn; Cu was significantly correlated with Mn, Ni, Pb, V and Zn; Ni was significantly correlated with V; Pb was significantly correlated with Zn; the coefficient were higher than 0.44 (p < 0.05).The intercorrelations of elements in Both IAP and RI were considered to assess the atmospheric environmental quality of urban Wuhan. As shown in p < 0.05; r < −0.4, p < 0.05; The appearance of IAP was somewhat in accordance with the spatial variation of RI. Sampling sites N15, N16, N19, N24 and N25 were found to be under low or moderate ecological risk, but they showed high IAP values, higher than 19 , suggestTerrestrial bryophytes have been widely used to monitor atmospheric depositions ,41. In tHylocomium splendens, H. cupressiforme, and P. schreberi, particularly abundant in European countries, such as Italy, Kosovo, and Poland [H. angustifolium, a pleurocarpous feather moss, which demonstrates epigeic and epiphytic inhabitation in the study region, was used to evaluate the atmospheric environment in our study. This species is widely distributed in subtropical humid climate to warm temperate and semi-humid regions  [The major criteria for selecting a suitable species appear to be its widespread distribution and large abundance in the study region to ensure the availability of adequate material for capturing contaminants and chemical analyses for multiple sites ,42. Howed Poland ,17,43, hl China)  and has l China) ,25,45.H. angustifolium is a good biomonitor of certain elements. Mosses can be excellent biomonitors for certain elements, such as Cd or Pb, probably because these elements almost exclusively originate from the atmosphere [Mosses appear to be good biomonitors of atmospheric trace elements, such as Cd, Pb, Cu, V, and partially Zn . In partmosphere . The mosmosphere . Pollutimosphere . In Wuhamosphere . As alsomosphere  in urbanSoil may be another source of trace elements accumulated in mosses. Although mosses do not take up substances directly from soil, local soil particles may strongly influence the chemical composition in mosses by blowing wind; the entrapped particles on the moss surfaces are then washed down and partly dissolved by precipitation, thereby enriching several elements in mosses ,49. OtheSeveral trace elements in moss samples showed significant correlations. For example, positive correlations between the concentrations of Cd and Pb in mosses were found in this study and in most European countries ,51; thisIAP, which was introduced by le Blanc and de Sloover , is one Generally, RI was significantly negatively correlated with IAP for the study sites, the spatial variation in RI was somehow negatively in accordance with IAP. Unlike the diversity index IAP, RI evaluates the contamination levels of several trace elements’ composition in biomonitors. For the RI of soil trace elements, approximately 72.4% of the urban areas in Wuhan have reached the level of considerable potential ecological risk (300 ≤ RI < 600) . SimilarH. angustifolium, which is a pleurocarpous feather moss, appeared to be a good biomonitor of atmospheric quality in central China. The urban areas in Wuhan generally faced a considerable potential ecological risk level caused by atmospheric trace element pollution, and Cd exhibited the highest ecological risk level among the 10 elements. This study suggests that effective measures should be taken into consideration to alleviate certain trace element contamination in the atmosphere and reduce the ecological risk level of trace elements in this major metropolis.The use of the diversity of bryophytes and the accumulated trace elements in them in urban areas of Wuhan City exhibited a clear spatial pattern of bryophyte functional richness and the concentrations of 10 elements . Environmental heterogeneity greatly influenced the occurrence and abundance of bryophytes in terms of varied life forms, coverage, and IAP. Among all of the species in the urban areas,


Irritable bowel syndrome (IBS) has a high morbidity rate worldwide, but there are no effective treatment measures, which seriously affect people's lives. Previous clinical studies on Tong-Xie-Yao-Fang (TXYF) combined with Si-Ni-San (SNS) in the treatment of IBS have been increasing, but there is no systematic evaluation. This study aims to systematically study the effectiveness of TXYF combined with SNS in the treatment of IBS.The PubMed, EMBASE, Science Network, Cochrane Library, Chinese Biomedical Literature, Wanfang Chinese Digital Journal and Conference Database, China National Knowledge Infrastructure Database and VIP China Science and Technology Journal Database (VIP) will be used Search related literature, and the search time is from the date of establishment to February 2021. The National Institutes of Health clinical registry Clinical Trials, International Clinical Trials Registry Platform and the Chinese clinical trial registration platform will be searched to find ongoing or unpublished trials. After screening the literature according to the criteria, two researchers independently extracted data according to a predetermined table. The primary outcome is total effective rate. The RevMan 5.3.5 software will be used for statistical analysis. Finally, the recommendation, evaluation, development and evaluation system will be used to evaluate the quality evidence for each result.This study will provide the latest evidence of efficacy for the TXYF combined with SNS for IBS.The effectiveness of TXYF combined with SNS for IBS will be evaluated.INPLASY202120075. According to epidemiological studies, the prevalence of IBS worldwide is about 5%-10%. The annual medical costs related to IBS in Europe are estimated to be about 8 billion euros, China is about 123 billion yuan, and the United States is about 10 billion or more.Irritable bowel syndrome (IBS) is a functional digestive system disease characterized by bowel habits, bloating, abdominal pain and changes in stool characteristics, without organic disease, and belongs to one of gastrointestinal neurosis. In patients with severe symptoms, low-dose tricyclic antidepressants, intestinal secretions, and antibiotics are often used.,9 Due to the lack of indicators such as bacteriological and biochemical abnormalities in IBS, there is no ideal cure drug and symptomatic treatment not only has limited efficacy, but also has a high recurrence rate. Based on the current situation, there is an urgent need to find an effective treatment for IBS.The main purpose of current treatment of IBS is to improve abdominal pain and bowel habits. Common treatment measures include changing eating habits, taking soluble fiber and antispasmodic drugs.,12 Tong-Xie-Yao-Fang (TXYF) and Si-Ni-San (SNS) are two ancient formulas that are widely used in the treatment of IBS in China.,14 TXYF can improve visceral hypersensitivity in rats, and its mechanism may be related to the up-regulation of TAGLN and Aldh2 and the down-regulation of CK8. Moreover, it can improve intestinal permeability and intestinal mucosal barrier function, which may be related to the inhibition of inflammatory cascade and NF-κB and Notch signaling pathway. The multifunctional synergistic effect of SNS on IBS, including inflammatory response regulation, oxidative stress suppression regulation, and hormone and immune regulation, which may be related to Prostaglandin G synthase-2, Calmodulin-2 and other protein.At present, complementary and alternative therapies are getting more and more attention from IBS patients and clinicians, including traditional Chinese medicine therapies represented by Chinese herbal medicine.In summary, the effectiveness of TXYF combined with SNS in the treatment of IBS is worth studying. Although there have been some randomized controlled trials (RCTs) of TXYF combined with SNS in the treatment of IBS, there is no evidence-based medicine to support it. Therefore, we will collect all relevant RCT data for meta-analysis to evaluate the efficacy of TXYF combined with SNS in the treatment of IBS and provide the best available evidence for it.2To evaluate the efficacy of TXYF combination with SNS, in people with IBS. And provide the latest evidences of evidence-based medicine for the clinical treatment of IBS.33.1https://inplasy.com/inplasy-2021-2-0075/). The protocol of our study will strictly follow the Preferred Reporting Items for Systematic Review and Meta-Analysis Protocols Statement Guidelines for systematic reviews and meta-analysis.The protocol has been registered by us on the INPLASY website  will be searched by us for relevant literature. We will search for data in the above 9 Chinese and English databases, and the search time will from their inception to February, 2021. The key words include “tongxieyaofang,” “sinisan,” “irritable bowel syndrome,” “Irritable Syndrome,” “IBS,” and “random allocation.” We will also search ongoing or unpublished trials from the National Institutes of Health clinical registry Clinical Trials, International Clinical Trials Registry Platform and the Chinese clinical trial registration platform. PubMed's search strategy is shown in Table 3.33.3.1All RCTs of TXYF combined with SNS in the treatment of IBS, whether blinded or unblinded.3.3.2All patients diagnosed with IBS based on specific diagnostic criteria .3.3.3The treatment of the experimental group is TXYF combined with SNS. The mode of administration is oral, and the dosage form is decoction or granule.3.3.4The control group was treated with conventional drugs or placebo.3.3.5The primary outcome measure is total effective rate. The secondary outcome measures include symptom score, functional digestive disorder quality of life scale, etc.3.4(1)Non-RCT literature.(2)Animal experiments, case reports and reviews, etc.(3)Repeatedly detected or published literature.(4)Secondary IBS caused by other underlying diseases(5)Unable to obtain complete data or full text literature.3.5Two researchers independently screened the literature. First, import the retrieved documents into EndNote X9 software to eliminate duplicate documents. Then the 2 researchers read the title and abstract, excluding obviously irrelevant documents. For uncertain documents, further read the full text to determine whether to include in the study. If there are inconsistent views, a third researcher will participate in the discussion and make the final decision. The selection process will be shown through the Preferred Reporting Items for Systematic Review and Meta-Analysis Protocols flow chart Fig. .In addition, two independent researchers extracted data from qualified literature according to a pre-designed data extraction table. The extracted content included the author's name, publication year, patient age, gender, diagnostic criteria, intervention measures, outcome indicators, and effect size.3.6 The risk assessment of bias includes seven aspects: random sequence generation; hidden grouping; blinding to the research objects and treatment plan implementers; blinding to the outcome measurers; incomplete data; selective reporting of research results; other biases. For each research result, make low-risk, high-risk and unclear judgments on the above 7 items. The quality evaluation is carried out independently and in parallel by 2 evaluators. In case of inconsistent evaluation results, they must be resolved after discussion.The Cochrane collaborative tools will be used to evaluate the quality of the literature.3.73.7.1The Review Manager (RevMan) V.5.3 software will be used for statistical analysis. When calculating the effect size, the relative risk and 95% confidence intervals are used for the dichotomous outcomes, and the weighted mean difference or standard mean difference will be used for the continuous outcomes.3.7.2I2 test were used to estimates the degree of heterogeneity. When I2 < 40%, it indicates that no significant statistical heterogeneity. When 40% < I2 < 75%, it indicates that statistical heterogeneity, and the source of heterogeneity needs to be further analyzed. When I2 > 75%, it indicates that there is statistics heterogeneity between the studies.Chi-square test was used to test the heterogeneity among the studies, and the test level was α = 0.1. 3.7.3Fewer than 10 studies do not require analysis of reporting bias. If more than 10 studies are included, we will use the symmetry of the funnel chart to detect potential reporting bias.3.7.4If there is significant heterogeneity between the research results, we will conduct a subgroup analysis to investigate the differences in age, gender, etc. And we will also use sensitivity analysis to evaluate the robustness of analysis results.3.7.5Two researchers used the Recommendation, Evaluation, Development, and Evaluation system to independently evaluate the quality of evidence for each result. The Grades of Recommendation, Assessment, Development, and Evaluation system divides the level of evidence into 4 levels: high, medium, low, and very low.3.8The study does not involve the personal information of participants, so no ethical approval is required. The research will be published in peer-reviewed journals or related journal meetings. The research results will provide new potential treatment strategies for patients with IBS.4,23 Although RCTs have shown that TXYF combined with SNS is effective in the treatment of IBS, there is no systematic evaluation and it has not been recognized internationally. Therefore, this study conducted a comprehensive systematic review and meta-analysis of the effectiveness of TXYF combined with SNS in the treatment of IBS, aiming to provide the latest evidence for the treatment of IBS and guide clinical decision-making.IBS has a high incidence worldwide, and there is still a lack of effective treatment measures. TXYF and SNS are used in the treatment of various gastrointestinal diseases in China. Studies have shown that they can regulate the intestinal flora, improve intestinal inflammation, etc.Conceptualization: Jiawang Jiang, Jing Ye.Data curation: Ziyi Hu, Huaiyu Li, Haiyi Tang.Formal analysis: Zhiying Yu, Yun Chen.Methodology: Haiyi Tang, Ziyi Hu, Yun Chen.Software: Zhiying Yu, Jiawang Jiang.Supervision: Jing Ye, Huaiyu Li.Writing – original draft: Jiawang Jiang, Huaiyu Li, Haiyi Tang.Writing – review & editing: Jiawang Jiang, Jing Ye, Haiyi Tang, Zhiying Yu.


With the change of people's life style, many more people are suffering from obese type 2 diabetes mellitus (T2DM). Acupoint catgut embedding is one of the acupuncture treatment principles in traditional Chinese medicine, which is widely used in the treatment of obese T2DM. However, there is no systematic review of the therapeutic effect of acupoint catgut embedding on obesity T2DM. Therefore, this article aims at the meta-analysis of acupoint catgut embedding in the treatment of obese T2DM, to clarify its curative effect.A structured and systemic literature search was conducted in the following databases up to December 1, 2019: PubMed, Cochrane Central Register of Controlled Trials (CENTRAL), Web of Science, EMBASE, CNKI, Wanfang Database. We will use the Review Manager 5.3 software provided by Cochrane collaborative network for statistical analysis. Then we assessed the quality and risk of the included studies and observed the outcome measures.This meta-analysis will further determine the beneficial efficacy of acupoint catgut embedding on obesity T2DM.The purpose of this meta-analysis is to explore the effect of acupoint catgut embedding intervention on obese T2DM patients, and provide more options for clinicians and patients to treat obese T2DM.This systemic review will evaluate the efficacy and safety of acupoint catgut embedding in the treatment of obesity T2DM. Since all the data included are published, the systematic review does not need ethical approval.CRD42020160801. Obesity is a major risk factor for the development of T2DM, as the associated chronic, low-grade, sterile inflammation contributes to both insulin resistance and β-cell failure. In the United Kingdom (UK), of the 3.2 million people with diabetes, an estimated 80% to 85% are overweight or obese, in whom weight gain could be potentially detrimental. More and more evidences suggest that weight loss has clinical significance in improving blood glucose control, lipid status, renal function, blood pressure, and quality of life for adults with obesity T2DM.–7 Currently, metabolic surgery is the most effective way to treat obesity T2DM.,9 However, there are some disadvantages in metabolic surgery, such as infection, dyspepsia, and high cost. Empirical data also suggest that mortality rates with metabolic operations are typically 0.1% to 0.5%. Thus, further treatment strategies, including integrated Chinese medicine, metabolic surgery, and routine pharmacology, are still urgently needed for obese T2DM.With the changes in diet and lifestyle, obesity has become an alarming health crisis of the 21st century. Catgut embedding acupuncture is an updated and improved form of classic manual acupuncture with the advantages of lowering expense and time, as well as longer lasting stimulation without additional biological effect in comparison with manual acupuncture. In recent years, Catgut embedding at acupoints is becoming much more popular in the treatment of obesity and diabetes. However, there is no systematic review of the therapeutic effect of acupoint catgut embedding on obesity T2DM. As we have seen, this is the first meta-analysis of RCT to explore the effect of acupoint catgut embedding intervention on obese T2DM patients.Acupoint catgut embedding is a treatment method originated from traditional acupuncture. For instance, the book “Magic Pivot, End and Beginning” has mentioned “those who have been ill for a long time, the evil spirit goes deep, and those who have been stabbing this disease will stay for a long time.”2 and the corresponding checklist used. This systematic review protocol was registered on the PROSPERO international registry of systematic reviews (ID: CRD42020160801).This protocol which has been reported is based on the Preferred Reporting Items for Systematic Reviews and Meta-Analyses Protocols (PRISMA-P) guideline2.12.1.12 which are diagnosed with T2DM will be included. The diagnosis of T2DM is in accordance with who's diagnostic criteria for diabetes in 1999. Meanwhile, the participants who are included have no limitation of age, sex, region, citizenship, and nationality. Cases related to serious diseases, pregnancy, and drug-induced obesity are excluded.Participants with standards of body mass index (BMI) ≥24 kg/m2.1.2Observation group: They received intervention using acupoint catgut embedding for a minimum duration of 4 weeks. The intervention method can be acupoint catgut embedding alone or acupoint catgut embedding combined with other therapies.Control group: Patients in the control group were treated with sham acupuncture, placebo, or hypoglycemic drugs. However, acupuncture combined with drugs will be excluded.2.1.3The primary outcome measures were fasting blood glucose, 2 hours postprandial blood glucose (2hG), glycosylated hemoglobin, and BMI. The second observation index mainly includes triglyceride (TG), cholesterol (TC), insulin secretion (INS), waist circumference, hip circumference, and so on. The adverse events as safety outcomes will be reported.2.1.4Randomized controlled clinical trials and quasi-randomized controlled trials will be included.2.2A structured and systemic literature search was conducted in the following databases up to December 1, 2019: PubMed, Cochrane Central Register of Controlled Trials (CENTRAL), Web of Science, EMBASE, CNKI, Wanfang Database. The language of the selected literature includes Chinese and English. The search terms include: obese T2DM; obesity and T2DM; acupoint catgut embedding; catgut embedding; catgut implantation.Searching other resources: The manual search mainly is used for searching relevant studies, such as “Chinese Journal of Endocrinology and Metabolism” before the database creation. Details of the selection process were shown in the flow chart and the screening process is summarized in a flow diagram Fig. .2.3At first, according to the inclusion and exclusion criteria, two researchers (WL and TC) independently selected the literature after reading the title and abstract. Secondly, by reading the full text, we exclude uncontrolled research, nonrandomized, inconsistent evaluation criteria and similar data. If any differences occur during the screening study, the third author (P-CL) would be intervened.2.4Two researchers (WL and TC) used a predesigned data extraction table to extract the data of the final included study, including author, year, sample size, course of treatment, intervention measures, outcome indicators, adverse reactions, among others. The process of study selection will be performed by using the methods according to the PRISMA guidelines, presenting in the flow diagram.2.5 Statistical heterogeneity analysis was carried out for the Clinical RCTs included. Cochrane I2 test was used. When I2 was <50% or P > .05, it indicated that there was no statistical heterogeneity between the studies, the fixed-effect model was selected to combine the effect amount; otherwise, the random effect model was adopted.We will use the Review Manager 5.3 software provided by Cochrane collaborative network for statistical analysis. For continuous variables, mean and SD of each study were obtained and pooled as mean difference (MD) or standardized mean differences (SMD) with a 95% confidence interval (CI).2.6The literature quality of this study was evaluated by the bias risk table proposed by Cochrane collaborative network. The risk table includes 6 items: random sequence generation mode, whether to use allocation concealment, whether to blind the subjects and intervention providers, whether to blind the results evaluators, whether the results data are complete, whether to select the results report and other bias sources. The criteria used to assess the risk of bias are “low risk,” “high risk,” and “unclear.” In this process, 2 evaluators independently evaluate the methodological quality. In case of disagreement, the third author would be intervened.2.7I2 < 50%) between a group of studies, the fixed-effect model was used for evaluation. If there is significant heterogeneity (I2 > 50%) between a group of studies, the random-effect model is used for evaluation, and then sensitivity analysis or subgroup analysis is conducted as required to explain the heterogeneity.If there was no significant heterogeneity 3 The rise in prevalence of obesity coincides with the prevalence of T2DM. It has been estimated that by the year 2040, a staggering 642 million people will suffer from this disease worldwide. In this connection, many doctors have explored many methods for the treatment of this disease, which has also been widely applied in clinical practice, such as oral medication, or surgical treatment. However, there are some deficiencies in these treatments. When the drugs are taken orally, there will be effects on the liver and kidney function of the human body, or when the drugs are stopped, the disease will be repeated. The risk of infection also exists in surgical treatment.In recent years, with the change of people's lifestyle and the increase of work pressure, the incidence of diabetes is increasing. In the occurrence and development of diabetes, there will be many complications, such as obesity. Obesity is not only its complication, but also its inducing factor. Insulin resistance is the common basis of obesity and T2DM.Traditional Chinese medicine believes that the formation of obese diabetes is mainly due to the irregular diet, excessive diet, fat, sweet, and thick taste, whereas the internal factors of the formation of the disease are the disorder of spleen and stomach and the accumulation of phlegm and dampness.It is recorded in the book of Treatise On strange diseases that “the fat make people hot inside, and the sweet make people full inside.” Obese people tend to have copious phlegm. Therefore, this kind of disease is mostly based on deficiency in origin and excess in superficiality. Acupoint catgut embedding therapy is an improved application of acupuncture and moxibustion. It can produce continuous physical stimulation on acupoints to achieve the therapeutic purpose. Meanwhile, Catgut Embedding at acupoints is also widely used in clinical practice.Traditional Chinese medicine research shows that acupuncture and moxibustion can play an auxiliary role in controlling blood sugar and reducing weight.However, there is no systematic meta-analysis of the therapeutic effect of Catgut Embedding at acupoints on obese T2DM. Therefore, we plan to make a systematic review of acupoint catgut embedding therapy for obese T2DM, so as to provide high-quality evidence for the effectiveness and safety of acupoint catgut embedding therapy for obese T2DM.4However, we cannot avoid the following problems when we collect literature: the quality of literature is not high enough; the literature only includes English and Chinese, which is not comprehensive enough; the sample size of the included literature is not large enough.Conceptualization: Chunli Piao, Qi ZhangData curation: Cheng Tang, Li WangData synthesis: Cheng Tang, Li WangMethodology: Chunli Piao, Qi ZhangSoftware: Chunli Piao, Qi ZhangWriting draft: Chunli Piao, Qi Zhang, Huiyan Fu


Extra osseous Ewing sarcoma (ES), an uncommon malignant neoplasm, accounts for about 15% of Ewing sarcoma, which mainly affects paravertebral region, lower extremity, chest wall, retroperitoneum, pelvis, and hip. Here is a 54-year-old woman of primary vaginal Ewing sarcoma with uterine fibroid, which has been fewly known or reported.The patient was admitted to our hospital because of vaginal pain. Her uterus showed as parallel position and enlarged as about 3 months of pregnancy size.Magnetic resonance imaging (MRI) and ultrasonography (US) demonstrated 2 heterogeneous masses in the vagina and uterus, respectively. Ultrasound-guided puncture biopsy revealed a malignant tumor in the right lateral vaginal wall.The patient was treated by hysterectomy, bilateral salpingo-oophorectomy, and tumors excision, with the subsequent treatment of chemotherapy.The patient recovered well without local recurrence for >1 year.Primary vaginal Ewing sarcoma is extremely rare. The treatments of uterine fibroid include uterine artery embolization and surgical options, While wide local excision followed by adjuvant chemotherapy and/or radiotherapy should be recommended for the vaginal ES. A 48-year literature review from 1970 to 2018 revealed that <30 cases of primary vaginal Ewing's sarcoma have been previously reported, from which few had discussed the imaging findings about primary vaginal Ewing sarcoma.Vaginal ES and uterine fibroid have certain manifestations and imaging features in MRI and US, but not specific.In addition, transvaginal ultrasound-guided puncture biopsy from the vaginal mass revealed the diagnosis of Ewing sarcoma.1 Extraosseous Ewing sarcoma (EES) grows rapidly, with round-cell malignancy of uncharacterized mesenchymal cell origin. Virtually, all Ewing sarcomas share a common chromosomal translocation of the long arm of chromosome 11 and 22. The translocation of the EWSR1 gene on to chromosome 22p12 next to the FLI1 gene may cause upregulation of insulin-like growth factor 1, playing a key role in cellular proliferation. The common translocation and a strong membranous expression of CD99 could unify the diagnosis of extraosseous Ewing sarcoma. A 48-year literature review from 1970 to 2018 revealed that <30 cases of primary vaginal Ewing's sarcoma had been previously reported,,2,4 and few literatures had discussed the imaging findings of primary vaginal Ewing sarcoma. In addition, uterine fibroid is the most commonly benign tumor in female pelvis, and occurs in about 20% to 50% of women around the world, from which black women of reproductive age have the highest incidence.–7 We present a rare case of a 54-year-old woman with primary vaginal Ewing sarcoma accompanying with uterine fibroid. Lesions were detected by ultrasonography (US) and magnetic resonance imaging (MRI) examination.As a highly malignant neoplasm of bones, Ewing sarcoma (ES) usually occurs during childhood, and nearly 15% of ES are extraosseous.2A 54-year-old woman was admitted to our hospital for the pain in the vagina. She had been with the symptom for about 4 days. Upon gynecological examination, the vaginal tumor had the following characteristics: painful mass, indurated and immobile, with the size of 6.5 × 6.0 × 4.0 cm. The mass lesion arised in the right lateral vaginal wall, extending to labia majora. Her uterus showed as parallel position and enlarged as about 3 months of pregnancy size. Cervix and all the vaginal fornices were free from mass.MRI images revealed a 7.5 × 4.9 cm solid and cystic mass, which was identified in the ischiorectal fossa adjacent to the right lateral vaginal wall. The mass had obscure margin, while lesion's edge showed both restricted diffusion and marked enhancement. However, the location of the mass was considered to be indeterminate on MRI. Additionally, the uterine mass, measured 6.7 × 4.8 cm, had smooth and well-defined margin, but with inhomogeneous enhancement Fig. A–E.US images showed a hypoechoic or anechoic mass in the right lateral vaginal wall, measured 6.72 × 4.66 cm, with no obvious blood flow signal Fig. . CT scan2, Etoposide 62.8 mg/m2). After 6 weeks of combination chemotherapy, there was a complete remission of the tumor by response evaluation criteria in solid tumors (RECIST) criteria. For >1-year follow-up, the ultrasound and other clinical examination demonstrated that the patient remained free of local recurrence and metastasis.Final surgical pathology of the 2 masses was confirmed as vaginal ES and uterine fibroid, respectively , cystic, calcified, myxomatous, lipomatous, and sarcomatous, and the degenerations are rarely pure occurring in fibroid.Ewing sarcoma (ES) and primitive neuroectodermal tumor (PNET) are considered as single tumor category known as Ewing family of tumors (EFTs), There have been less case reports describing the imaging appearance of vaginal ES. On MRI examination, the tumors may be T2 isointense to hyperintense, T1 isointense to muscle, with heterogeneous contrast enhancement, while the tumor could demonstrate a heterogeneous ultrasonic appearance with few Doppler color flow. During ultrasound examination, fibroids usually appear as well-defined, solid, concentric, hypoechoic masses, with a variable amount of acoustic shadowing. MRI and computer tomography might be helpful in the diagnosis of uterine fibroid. A nondegenerated fibroid is usually as isointense to myometrium on T1 and hypointense on T2 sequences, and shows avid enhancement in MRI scans. Furthermore, different forms of degenerations may occur simultaneously in a given fibroid, thus the categorization on imaging may be based on the predominant imaging feature.The imaging findings of extraosseous ES is usually presented as a hypervascular soft tissue mass with areas of necrosis or hemorrhage. The tumors may also invade adjacent bones, but do not affect the bone marrow. Calcifications could appear in up to 25% of cases.Our case demonstrates a solid and cystic mass with obscure margin of the vaginal ES, while lesion's edge showed both restricted diffusion and marked enhancement in MRI images. Similar heterogeneous characteristics were seen on US, which manifested as a hypoechoic or anechoic mass, with no obvious blood flow signal inside. The MRI and US features could be interpreted by extensive area of necrosis or hemorrhage, which are the pathological features of vaginal ES. On MRI, the uterine fibroid of our case had smooth, well-defined margin, and heterogeneous enhancement, with a little contrast agent entering the mass, which may be owing to tumor degeneration.–17 On MRI, the proposed diagnostic imaging criteria for extraskeletal ES relied on the absence of adjacent bony invasion. Our case had no bone invasion, but the location of vaginal mass was considered to be indeterminate on MRI. In contrast, US could present the accurate location of the vaginal tumor. In comparison with MRI, US has several advantages, as it is widely available, well tolerated for patients, and it could be combined with a ultrasound-guided puncture biopsy, which is a safe, fast, and reliable method for pathological diagnosis.Differential diagnosis for a patient with an aggressive pelvic (extraovarian) lesion should include rhabdomyosarcoma, extraskeletal ES, synovial sarcoma, malignant melanoma, and some less frequently carcinoma. A standard treatment guidelines available for vaginal ES is lacking due to rarity of its occurrence.,20 Wide local excision followed by adjuvant chemotherapy and/or radiotherapy is recommended. The outcome for the tumor is primarily dependent on the presence of distant metastases. In the absence of detectable metastases, 70% of children and adolescents are cured with a combination of intensive, multiagent chemotherapy with surgery and/or radiotherapy.,21 As to the present case, the patient was treated by hysterectomy, bilateral salpingo-oophorectomy and tumors excision, with the subsequent treatment of chemotherapy. The patient had remained free of local recurrence and metastasis for >1-year. Given the possibility of local recurrence, it is essential to have continuous close follow-up of the patient.Most uterine fibroids do not require treatment unless they are causing symptoms. After menopause, fibroids shrink, and it is unusual for them to cause problems. In those who have symptoms, uterine artery embolization and surgical options have similar outcomes with respect to satisfaction.In conclusion, we report an extremely rare case of vaginal ES with uterine fibroid. Imaging is crucial for defining the extent of disease, evaluating response to treatment, and detecting recurrent/metastatic disease. Vaginal ES and uterine fibroid have certain imaging features in MRI and US, but not specific, while ultrasound-guided puncture biopsy plays a key role in confirming the diagnosis of vaginal ES.The protocol for the present retrospective study was approved by the Ethics Committee of Hubei Cancer Hospital, and informed consent was obtained from the patient.Conceptualization: Gaolong Weng, Fang Li.Investigation: Shue Zeng, Hongbin Wang.Writing – original draft: Maolin Xu, Fang Li.Writing – review & editing: Yulin Liu, Shue Zeng.


The chronic complications of diabetes mellitus (DM) are accompanied by inflammatory manifestations. Our study aimed to evaluate a possible association between the inflammatory status (reflected by serum chitotriosidase and neopterin) and the timely evolution and occurrence of chronic microvascular complications in patients with type 1 DM. This observational, cross-sectional study included 82 type 1 DM patients from the Centre for Diabetes, Nutrition and Metabolic Diseases, Cluj-Napoca, Romania. Our results demonstrated a link between the extent of inflammation, evaluated by the enzymatic activity of circulating chitotriosidase, and the onset of microvascular complications, especially diabetic neuropathy and retinopathy. Chitotriosidase enzymatic activity showed an ascending evolution over time. In non-smoking patients, the increase in chitotriosidase activity was correlated with the extent of microalbuminuria and the decline of glomerular filtration rate, while in smokers, only the presence of a positive correlation between chitotriosidase activity and disease progression was noticed. According to our results, the time span between the moment of diagnosis and the onset of microvascular complications was longer in non-smokers than in smokers. These results also imply that increased chitotriosidase activity may be a predictor of endothelial dysfunction in type 1 DM. Diabetes mellitus (DM) is increasingly considered a major public healthcare problem. Currently, 463 million people worldwide are affected by this disease. The International Diabetes Federation (IDF) estimates that, by 2045, the number of patients with DM may reach 700 million. In Europe, the latest estimates show that 59.3 million people are affected, of whom 286.000 are children and adolescents aged 0–19 years) with type 1 DM. In Romania, the prevalence of this disease has reached 8.8%, with approximately 1 in 11 people being diagnosed with DM [9 years wChronic complications of DM affect multiple organs and systems and are largely responsible for the morbidity and mortality associated with this disease. The risk of chronic complications is directly proportional to the increased level of blood glucose, and their onset is usually detected after 10 years of persistent hyperglycaemia . The conDM is associated with an accelerated evolution of atherosclerosis, affecting all blood vessels, but especially those of the heart, brain and extremities. Owing to its specific pathophysiology, diabetic retinopathy is the first cause of blindness in patients aged 20–74 years. Likewise, the first cause of chronic kidney disease is diabetic nephropathy .DM develops as a result of a combination between insulin resistance and pancreatic beta (β) cell failure, which are aggravated by oxidative stress, endoplasmic reticulum stress, lipotoxicity and glucotoxicity. These cellular alterations induce an inflammatory response that plays an active role in the pathophysiology of DM. In response to environmental stress, macrophages, endothelial cells and adipocytes release inflammatory cytokines. Some of them are represented by tumour necrosis factor alpha (TNF-α), interleukin 1 (IL-1), interleukin 6 (IL-6) and chemokines, such as monocyte chemoattractant protein-1 (MCP-1) and interleukin 8 (IL-8). Their combined effects trigger the release of acute phase markers  and the accumulation of pancreatic amyloid .Two markers of macrophage activation, namely, chitotriosidase and neopterin, play an important part in the activation of immunological and inflammatory response. Currently, their activity is still the focus of intensive debate, as the exact mechanism by which they regulate these functions is poorly understood, and there is controversy regarding their protective or harmful effects .Among chitinases, human chitotriosidase is the best characterized enzyme, both from a clinical and biological point of view. Proinflammatory cytokines, such as granulocyte macrophage colony stimulating factor (GM-CSF),TNF-α, lipopolysaccharides (LPS), bacterial muramyl dipeptide and prolactin stimulate the expression of chitotriosidase in monocyte-derived macrophages, while interferon gamma (IFN-γ) and IL-4 inhibit the expression of chitotriosidase ,9,10.Elevated serum levels have been described in many pathologies involving immune system dysfunctions ,9. BesidNeopterin belongs to the class of pteridines and is derived from guanosine triphosphate (GTP). It is increasingly recognized as an independent marker of immune activation and an important predictive marker of cardiovascular risk. Human monocytes or macrophages appear to be the most relevant source of neopterin and 7,8-dihydrononeopterin. Cytokines, such as IL-2, TNF-α, LPS, GM-CSF, human immunodeficiency virus (HIV) and other viruses, may stimulate the release of interferon gamma (IFN-γ) from T cells and, thus, trigger the release of neopterin . ElevateOur study aimed to evaluate a possible association between the inflammatory status, reflected by circulating chitotriosidase and neopterin levels and the occurrence of chronic microvascular complications in patients with type 1 DM. We also sought to investigate any possible relationship between the studied biomarkers and the length of disease evolution until the occurrence of chronic complications.This observational cross-sectional study included 82 patients with type 1 DM, consecutively selected from the Centre for Diabetes, Nutrition and Metabolic Diseases, Cluj-Napoca, Romania, between January 2018 and November 2019. The diagnosis of DM and its chronic complications were established according to the criteria of the American Diabetes Association . PatientThe research protocol was approved by the Ethics Committee of the University of Medicine and Pharmacy Cluj-Napoca, Romania (no. 408/08.11.2017), in accordance with the revised Helsinki Declaration of 2000. Before enrolment, all selected patients signed an informed written consent form.Data collection was performed using patient medical records, for the following items: age and gender, height, abdominal circumference, weight and body mass index (BMI), time span since the diagnosis of DM, fasting glycaemia and HbA1c values, complete blood count indices and smoking status. We also recorded the eventual presence and type of chronic microvascular complications , the coexistence of arterial hypertension and of any other associated chronic disorders. Serum chitotriosidase enzymatic activity was measured by a fluorometric method, using an artificial substrate   tests were subsequently used for data analysis. Because of the highly dispersed values of quantitative variables that did not display a normal distribution, we used the Spearman correlation coefficient and its significance test. Quantitative variables were presented as means and standard deviations, or as medians and quartiles. For the description of qualitative variables, we used frequency tables and employed contingency tables to analyse their potential associations. For a better understanding of our approach, we chose to present the mean and standard deviation, even for variables that were not normally distributed.Our study comprised 82 patients with type 1 DM, of whom 44 (53.66%) were men. Neuropathy was present in 58.54% and retinopathy in 60.98% of patients. Microalbuminuria was present as follows: 75.61% had stage A1, 19.51% stage A2 and 4.88% stage A3. Only seven (15.9%) male and eight (21.05%) female patients received treatment with insulin by continuous subcutaneous infusion (insulin pump), the others being treated with insulin in basal-bolus regimen.9 platelets/L (±61.94) and a number of 7.03 × 109 leukocytes/L (±2.13). Smoking status was found in 12 (14.63%) male patients and in 10 (12.20%) female patients. Among the associated pathologies, chronic inflammatory diseases were present in 23.17% of patients.The mean age of the patients was 44 years (±13.39), with a body mass index (BMI) of 25.65 kg/m² (±4.37). The mean value of HbA1c was 8.85% (±1.78), and the mean duration of diabetes was 20 years . The mean glomerular filtration rate was 97.27 mL/min/1.73 m² (±20.68), the median UACR was 10.86 mg/g , the mean fasting glycaemia at diagnosis was 173 mg/dL , the mean insulin dose per kilogram body weight was 0.71 IU/kg (±0.26), and the mean value of LDL cholesterol was 107.49 mg/dL (±33.33). The median value of serum chitotriosidase activity was 147.5 nmol/mL/h , and that of neopterin was 9.31 ng/mL . Full blood count showed a mean haemoglobin value of 14.27 g/dL (±1.8), a number of 262.4 × 10No significant differences in age, disease duration, HbA1c, BMI, serum chitotriosidase or neopterin levels were noticed, according to neither gender distribution  nor micrFor the whole patient cohort, we found statistically significant differences between the enzymatic activity of chitotriosidase in patients with neuropathy and retinopathy, compared to those without these complications. However, no significant differences in chitotriosidase activity were observed among patients with microalbuminuria, regardless of its evolutionary stage. For the serum level of neopterin, no statistically significant results were obtained for any of the existing microvascular complications .For the entire patient cohort, a statistically significant difference between the enzymatic activity of chitotriosidase in smoking and non-smoking subjects was noticed. In the case of neopterin, the statistical significance was not maintained. From a statistical point of view, coexisting inflammatory diseases did not influence the values of studied biomarkers .Within the subgroup of patients with confounding factors, no statistically significant differences in chitotriosidase activity or neopterin levels were observed between the subjects with different types of chronic complications and those without complications .p < 0.001).The enzymatic activity of chitotriosidase in non-smoking patients has reached the threshold of statistical significance for neuropathy and retinopathy, but not for microalbuminuria. For neopterin, no statistically significant differences were observed for any of the chronic complications . When coOf the two biomarkers of macrophage activation, only chitotriosidase was positively correlated with the duration of DM. Neither of the two biomarkers reached statistical significance in terms of correlation with HbA1c or with the therapeutic insulin dose .A moderate, positive, statistically significant correlation was observed in both smoking and non-smoking patients between chitotriosidase enzymatic activity and disease duration. In non-smokers, both the age of the patients and the UACR were positively and significantly correlated with the inflammatory biomarker. Another correlation that reached statistical significance in non-smokers was a negative one, between chitotriosidase activity and the glomerular filtration rate (GFR) .2) [Our study aimed to evaluate the association between chronic inflammation and microvascular complications in patients with type 1 DM. Given that, on average, our patients had an increased percentage of HbA1c 8.85%), a long-term evolution of DM (20 years) and were mostly overweight , also called YKL-40, have been studied mainly in patients with type 2 DM. Previous studies have shown that in both type 1 and 2 DM, chitotriosidase activity was correlated with age, GFR and UACR, but not with glycaemia or HbA1c ,23,24,25Among chronic microvascular complications, neuropathy is the most common. Due to the lack of specific symptoms, its diagnosis can be easily overlooked, especially in the early stages of evolution. Our study has shown a considerable increase in serum chitotriosidase activity in patients with neuropathy, compared to those without it. The difference became even more important if neuropathy was associated with long-term disease duration . These results are consistent with those reported in patients with type 2 DM, affected by this complication .For the evaluation of circulating neopterin, we considered the reference range suggested by Nazar et al. . DespiteAfter 20 years of evolution, most DM patients have some degree of retinal damage, which is the most common cause of blindness . In thisThere is clear evidence that the pathogenesis of diabetic nephropathy is multifactorial, but inflammation plays a key pathophysiological role . After 1Because 26.83% of our patients were smokers and 23.17% had other chronic inflammatory diseases, we investigated these two variables as potential confounders. Chitotriosidase activity was undoubtedly higher in smokers, compared to non-smokers, with a statistically significant difference between the two patient subgroups. We presume that this result may be explained by the persistent inflammatory response generated by smoking ,35.According to our results, smoking and coexisting inflammatory diseases could mask the evolution of DM complications. Therefore, in these patients, no statistically significant differences were observed between the levels of inflammatory markers in subjects with and without chronic complications, although chitotriosidase had higher values in association with neuropathy. In the absence of confounding factors, significant differences were obvious for neuropathy and retinopathy, but not for microalbuminuria.Our study showed a statistically significant correlation between chitotriosidase values and the duration of the disease for all patients and in both subgroups (smokers and non-smokers). We only found a correlation between chitotriosidase activity and age in non-smokers, while in smokers it disappeared, due to the smoking-induced activation of the enzyme. Unlike the Ramanathan study , which sTo the best of our knowledge, this is the first evaluation of chitotriosidase as an inflammatory marker in patients with type 1 DM and microvascular complications. We demonstrated a link between the extent of inflammatory status, quantified by the enzymatic activity of chitotriosidase, and the occurrence of neuropathy and retinopathy in type 1 DM. Circulating chitotriosidase showed an increased progression over time. In non-smoking patients, its evolution was correlated with the aggravation of microalbuminuria and the decline of GFR, while in smokers, only a positive correlation between chitotriosidase activity and disease progression was observed. Moreover, according to our results, the length of disease evolution between the moment of diagnosis and the occurrence of microvascular complications was longer in non-smokers than in smokers, highlighting the impact of chronic inflammation on the progression of DM. In our study, circulating levels of neopterin were not associated with any of the microvascular complications in patients with type 1 diabetes.


Compared with the high-cereal concentrate, feeding the concentrate including the byproducts resulted in a reduction of potentially human-edible ingredients from 64.4% to 38.7%. In conclusion, 44% of cereal grains and protein feeds in the concentrate for light lambs can be replaced with a mixture of corn distiller’s dried grains with solubles, dried citrus pulp, and exhausted olive cake without negatively affecting growing performance and animal health.Feeding agroindustrial byproducts to ruminants can have multiple benefits, such as lowering feeding costs, reducing competition with human food, decreasing environmental impact associated with byproducts disposal, and improving the quality of animal products. In order to use these byproducts in practical feeding, their effects on animal performance and health should be assessed. In this study, we evaluated the effect of replacing 44% of conventional feeds in a high-cereal concentrate for light lambs with three byproducts: Corn distiller’s dried grains with solubles (18%), dried citrus pulp (18%), and exhausted olive cake (8%), all of which are highly produced in the Mediterranean area. We observed that the inclusion of these byproducts did not affect feed intake, growth performance, ruminal fermentation  dry matter digestibility. Diet had no effect on post-mortem ruminal pH and total volatile fatty acid concentrations and profile, but NH3-N concentrations were lower (p = 0.003) for the BYP-fed group compared with the control one. However, plasma concentrations of amino acids, total proteins, urea, and hepatic enzymes were not affected by the diet. In conclusion, 44% of feed ingredients in the concentrate for light lambs can be replaced with a mixture of corn DDGS, DCP, and EOC without negatively affecting growing performance and animal health.The objective of this study was to evaluate the effect of replacing cereals and protein concentrates in a high-cereal concentrate (control) for light lambs with corn distiller’s dried grains with solubles , dried citrus pulp , and exhausted olive cake  in a byproduct (BYP) concentrate on growth performance, digestibility, ruminal fermentation, and plasma metabolites. Two homogeneous groups of Lacaune lambs (13.8 kg ± 0.25 kg) were fed one of each concentrates and barley straw  Agroindustrial byproducts have been traditionally used for small ruminant feeding in low-input systems, but their use is continuously increasing worldwide. Although either the shortage or the high cost of conventional feeds has been the main reason for feeding byproducts to livestock, other reasons are equally or even more important to support their current use. Some byproducts are highly contaminant, and their use in animal feeding can help to reduce the environmental problems caused by their accumulation and to lower the carbon footprint of animal products when locally produced byproducts are used . Most by6 t for the 2019/20 crop year [Three byproducts originated in the olive oil, citrus, and ethanol industries, all highly produced in the Mediterranean area, were used in this study. Olive oil production has tripled in the last 60 years with an estimated production of 3.1 × 10rop year  and is crop year . Citrus rop year . The mairop year ,9,10, anThe lambs used in this trial were cared for and handled in accordance with the Spanish guidelines for experimental animal protection, and experimental procedures were approved by the General Direction of Livestock and Agriculture of the Community of Madrid .ad libitum concentrate and barley straw and had free access to fresh water over the trial. The barley straw contained (as-fed basis) 92.3%, 7.25%, 2.90%, 1.60%, 71.9%, and 38.0% of dry matter (DM), ashes, CP, ether extract (EE), NDF, and acid-detergent fiber (ADF), respectively. According to INRA [Twenty four Lacaune male lambs, with an initial body weight (BW) of 13.8 ± 0.25 kg, were homogeneously distributed in two groups according to their BW. Lambs were penned individually in 1-m x 1-m pens with slatted floor, which were placed 1 m above the floor and equipped with two feeders and an automatic drinker. Each group of lambs was randomly assigned to one of the two dietary treatments: A high-cereal concentrate (control) and a byproduct containing concentrate (BYP), in which 44% of conventional feed ingredients  were replaced with corn DDGS, DCP, and EOC . The ingredients and chemical composition of both concentrates is shown in  to INRA , 1 kg ofg, 10 min, 4 °C), and the plasma was immediately frozen (−20 °C) until determination of concentrations of amino acids, albumin, globulins, total proteins, urea, and cholesterol, the activities of the enzymes lactate dehydrogenase (LDH), alkaline phosphatase (ALP), creatine phosphokinase (CPK), glutamic oxaloacetic transaminase (GOT), glutamic pyruvic transaminase (GPT), and gamma-glutamyl transpeptidase (GGT).The experiment lasted for six weeks and included seven days for diet adaptation. Concentrate and straw intakes were measured twice per week, whereas BW of all lambs was determined weekly. Samples of offered concentrate and straw were taken weekly for analysis of chemical composition. On days 0, 21, and the slaughter day, blood samples were taken from each lamb by jugular venipuncture into tubes containing EDTA immediately before feeding. Samples were centrifuged  to keep the pH below 3.0 [In the fourth week of the trial, the digestibility of the diets and the nitrogen balance were measured in nine lambs per treatment. Trays were placed under the slated floor of each pen for feces and urine collection. The trays had holes for urine collection, which was collected in a bucket containing a solution of Helow 3.0 . The fec3-N concentrations. In addition, the color of the rumen epithelium was evaluated as described by Haro [In the last week of the trial, lambs were slaughtered at a commercial slaughterhouse located 20 km away from the experimental farm on two different days. The six lambs of each treatment with the greatest body weight were slaughtered the first day, and the rest of lambs the second day. Lambs had free access to feed and water until about 2 h before slaughter, and were slaughtered according to commercial practices involving head electrical stunning and severing the carotid arteries and jugular veins. After slaughter and dressing, the full gastrointestinal tract was removed and samples from rumen contents were immediately taken. The ruminal content was homogenized, a sample of about 300 g was filtered through four layers of gauze, and the pH of the fluid was immediately measured using a Crisson Basic 20 pH-meter . Then, 2 mL of fluid were mixed with 2 mL of 0.5 N HCl and samples were frozen (−20°C) until analyses of volatile fatty acids (VFA) and NH by Haro . Briefly3-N concentrations were determined by the phenol-hypochlorite method of Wheatherburn [The procedures of the Association of Official Analytical Chemists  were usetherburn .Plasma concentrations of amino acids were determined by high-performance liquid chromatography (HPLC) as described by Frank and Powers , whereasp < 0.05, and p values between 0.05 and 0.10 were considered trends.Data on feed intake, growth performance, ruminal fermentation, meat composition, digestibility, and N balance were analyzed with one-way analysis of variance using the GLM PROC of the SAS . Data onThe proportion of each byproduct in the diet was selected from the results of in vitro studies by our group  and publAs shown in p ≥ 0.704) or carcass yield compared with control concentrate. Values for these parameters were in the range previously reported by others for light lambs fed high-cereal concentrates and slaughtered at about 25–26 kg BW [There were no differences between groups in final BW, average daily gain (ADG), and feed conversion rate. In agreement with these results, feeding the BYP concentrate did not affect carcass weights  for control than for BYP concentrate (p ≥ 0.120) between concentrates in the digestibility of CP, NDF, and ADF. The lower DM and OM digestibility of the BYP concentrate may have been partly due to its lower content in non-structural carbohydrates , which is a highly digestible fraction, and its greater lignin content (1.79% vs. 2.80%). Values of diet digestibility were in the range reported by others for lambs fed high-cereal diets [The digestibility of both DM and organic matter (OM) was greater  N intake of the BYP-fed lambs was due to the greater CP content of BYP compared with the control concentrate, as there was no differences between groups in concentrate intake (p = 0.012) than that for control group, but differences disappeared when fecal N excretion was expressed as proportion of the ingested N, which is consistent with the lack of differences in CP digestibility. There were no differences between groups in the amount of urinary and retained N, either expressed as g/d or as a proportion of the ingested N. Values of N retention were in the range of those previously reported for young growing lambs fed diets with similar CP levels [The greater (e intake . The daie p = 0.0 than thaP levels .p ≥ 0.247) of the type of concentrate on post-mortem ruminal pH, total VFA concentrations, molar proportions of individual VFA, or acetate/propionate ratio (p = 0.003) NH3-N concentrations in the rumen of BYP-fed lambs compared with control-fed lambs might indicate lower CP degradability in the BYP-concentrate. In fact, DDGS protein has lower ruminal degradability than other concentrate feeds, such as soybean and palm meal [3-N in the rumen of control-fed lambs were adequate for microbial growth [There was no effect  to darker color of the ruminal epithelium in the lambs fed the BYP-concentrate are unclear. A darker color of ruminal epithelium is usually associated to keratinized tissue [The reasons for the trend  of the diet on plasma concentrations of any amino acid, and no diet x sampling day interactions (p ≥ 0.151) were detected (3-N concentrations. In agreement with previous studies [p ≤ 0.065) to increase as lambs grew older, with the exception of glutamic acid, histidine, glycine, arginine, alanine, and cysteine, which did not change over the trial. The increased amino acids concentrations at the end of the trial are in accordance with the greater growth rate of the lambs at this time, as amino acids are used for protein synthesis and tissue formation [There were no effects  were detected in any of the metabolites measured in the blood of lambs (p = 0.585) is in accordance with the similar concentrations of amino acids observed in both groups of lambs. Plasma urea concentrations reflect the amount of urea produced in the liver from amino acid catabolism [3-N absorbed from the rumen. The similar (p ≥ 0.276) levels of albumin, globulins, and total proteins in the plasma of both groups agree well with the lack of differences in plasma concentrations of amino acids and urea. Plasma concentrations of these metabolites, excluding globulins, increased with time. The normal [No diet x time interactions (of lambs . The lactabolism , althouge normal ,40,41 anFinally, it is worth mentioning that under the conditions of our study, the cost of the control and BYP concentrates were 0.270 €/kg and 0.262 €/kg, respectively, resulting in a calculated cost of concentrate per kg of gain was 0.787 € and 0.777 € per control and BYP concentrate, respectively. However, these results may vary, as feed prices are highly volatile.Our results indicate that a mixture of corn DDGS, DCP and EOC can replace 44% of cereal grains and protein feeds in the concentrate for light lambs without negatively affecting the feed intake, growth performance, or health of lambs. The use of these byproducts reduced the potentially human-edible ingredients in the concentrate from 64.4% to 38.7% and slightly decreased the feeding costs. In situations of high prices of cereals and protein feeds, the use of byproducts by lamb farmers can be more economically profitable than in the present study.


The development of a colorimetric mono-varietal discriminating assay, aimed at improving traceability and quality control checks of durum wheat products, is described. A single nucleotide polymorphism (SNP) was identified as a reliable marker for wheat varietal discrimination, and a rapid test for easy and clear identification of specific wheat varieties was developed. Notably, an approach based on the loop-mediated isothermal amplification reaction (LAMP) as an SNP discrimination tool, in combination with naked-eye visualization of the results, was designed and optimized. Our assay was proven to be effective in the detection of adulterated food products, including both substitution and mixing with different crop varieties. Triticum aestivum species is considered a contamination that must not exceed the 3% maximum level [Food traceability currently has a key role in the agrifood sector, bringing many benefits to both industries and consumers, such as supply chain optimization, improvement in food safety and quality, and an increase in controls and in the consumers’ confidence. Mandatory traceability regulations have been established by several countries with different level of stringency for both domestic and imported commodities and agrifood products  of wheat variety to guarantee plant breeders’ rights (PBR). Distinctness, uniformity, and stability are among the basic requirements for a variety. The traditional morpho-physiological descriptors, collected during the whole plant life cycle, are classically used for such evaluation purposes. In a further step, morphological descriptors have been flanked by biochemical markers, e.g., the isoforms of seed storage proteins. Such markers can be informative when applied to the grains, but are not effective on transformed products, in which technological processes can impair the proteins’ integrity. The subsequent advancement in varietal fingerprinting was based on the introduction of DNA markers that are independent of the environmental influence and available in theoretically unlimited numbers. Many classes of DNA molecular markers have been developed, ranging from inter simple sequence repeats (ISSRs) to amplified fragment length polymorphism markers (AFLP) and random amplified polymorphic DNA (RAPD), as reviewed by Pasqualone, 2011 . SSRs , was included in the analysis. All of the varieties were certified foundation seeds produced directly from the breeder responsible for their maintenance in purity. Ten certified seeds of each variety were sown in duplicates in pots, and the foliar tissues were harvested at the three-leaves stage. DNA samples were isolated and purified using the cetyl trimethyl ammonium bromide method [The following 28 durum wheat varieties were used: Iride, Rusticano, Saragolla, Odisseo, Maestrale, Bronte, Antalis, Fabulis, Core, Svevo, Orizzonte, Aureo, Achille, Monastir, Claudio, Tirex, Pigreco, Normanno, Marco Aurelio, Relief, Miradoux, Babylone, Simeto, Anvergur, Navigator, Levante, Kyle, and Kronos. Moreover, the tetraploid e method . The quahttp://www.diversityarrays.com/, Canberra, Australia) for sequencing, and SNP marker identification was done by DArTseq genotyping. Sequences of the genomic representations were obtained on a HiSeq2500 instrument.Purified DNA samples (1 μg for each sample) were sent to Diversity Arrays Technology Pty Ltd. . SNPs with low frequent alleles were kept in the dataset to allow the research of private alleles of Aureo. This resulted in a dataset containing 20,198 SNPs and 58 samples (two genotyping replicates for each variety). After marker curation, all samples had a percentage of missing values below 10% and a percentage of heterozygous calls below 12%. Genetic distances were calculated based on simple matching.A custom R script was used to select SNPs with no missing values and a private allele of both replicates of Aureo. Candidate SNPs were mapped on the durum wheat genome sequence  by BLASTLAMP primers were designed from the 7A chromosome of durum wheat. Primer sequences are shown in 4)2SO4, 20 mM of MgSO4, 500 mM of KCl, and 1% v/v Tween 20), 2 mM of MgSO4, 0.8 mM each of dNTPs , 0.112 U/μL of Bst 2.0 WarmStart DNA Polymerase , and 5 μL of DNA template at the concentration of 5 ng. DNA-free LAMP reactions were included as negative controls. Amplification reactions were performed using forward and backward inner primers (FIP and BIP), forward and backward outer primers (F3 and B3), and one loop primer (LoopT or LoopG). For real-time fluorescent LAMP, 1/50,000 diluted SYBR Green  was pre-added to the reaction mix. Real-time amplifications were performed on an Applied Biosystem real-time instrument  with StepOne Software v2.3 at 63 °C.LAMP reactions were performed in 25 μL of a mixture containing 1.6 μM each of inner primers, 0.4 μM each of outer primers, 0.8 μM of loop primers , 1 M of betaine , 2.5 μL of 10× LAMP buffer 2SO4, 20 mM of MgSO4, 500 mM of KCl, and 1% v/v Tween 20), 2 mM of MgSO4, 0.8 mM each of dNTPs , 0.112 U/μL of Bst 2.0 WarmStart DNA Polymerase , and 5 μL of DNA template at the concentration of 5 ng. DNA-free LAMP reactions were included as negative controls. For visualized detection, 0.06 mM of Cresol Red  was pre-added to the reaction mix. The amplification efficiency was verified by color changing of the reaction mix. Colorimetric LAMP reactions were performed on a T100 Thermal Cycler  at 63 °C for 1 h, followed by heat inactivation at 90 °C for 2 min.LAMP colorimetric reactions were performed as reported above, using 1 M of betaine , 2.5 μL of 10× LAMP buffer . In order to develop a single-SNP assay, we selected SNPs tagging a private allele of Aureo. The genotyping of two replicates per variety reduced the risk of selecting a private SNP allele caused by a genotyping error. The average genetic distance between two replicates was 98.5% (simple matching), suggesting a genotyping error rate of 1.5% on average. A set of 11 candidate SNPs carrying a private allele of Aureo was identified, with six of them presenting no missing values. To ensure the selection of a private SNP allele, the SNP marker to be used for development of the LAMP assay was selected among those six SNPs with complete genotypes. The assay developed in this study was based on an SNP located on Chr 7A.The low missing rate and the number of SNPs generated in this study enabled the identification of six SNPs as potential candidates for the assay. This was sufficient for developing our single-SNP approach, which aimed at the discrimination of a variety within a relatively small panel. Such an approach would be very suitable for specific industry requirements.Such a need is relevant in several high-value agrifood products. In Italy, for example, some PGI  products, such as Amarene brusche di Modena, Ciliegia di Vignola, and Pera dell’Emilia-Romagna, are regulated by product specifications that require the use of specific varieties. Moreover, worldwide wine production is linked to the use of specific grape varieties. Various wine laws include appellation-based regulations that cover not only the area of production and the wine-making practices, but even the permitted grape varieties. European PDO (protected designation of origin) and PGI are examples of such regulations directed to guarantee that a certain wine is made from a percentage of grapes belonging to a specific variety or to a specific panel of varieties. However, the genotyping effort required to identify a private SNP allele might be higher in situations with a larger panel of varieties or lower genetic diversity. To overcome this issue, a multi-SNP assay could be developed, since a private allele can be tagged more easily using more than one SNP.In this framework, LAMP has been recently proven to be an interesting tool to achieve SNP genotyping . In geneIn this work, we optimized this approach for Aureo varietal discrimination, exploiting the high tolerance of LAMP to contaminants and the possibility of instrument-free visualization of the results through pH-sensitive dyes ,28, whicThe design of loop primers has a pivotal role in this strategy. In particular, the approach relies on the stabilization of the annealing between short loop primers and the perfectly complementary sequence, while the non-specific one is disfavored. Loop primers act as accelerating primers, speeding up the onset of the amplification. Hence, the reaction in which the loop primer is perfectly hybridized will be properly enhanced, unlike the one with the unstable annealing of the primer, which will be delayed . A singlThis strategy was applied to the assessment of a wide range of different wheat varieties to analyze the reliability of the technique in a real scenario of quality control. Interestingly, the method was able to efficiently discriminate all of the 13 tested varieties of wheat versus Aureo , with anOnce we assessed that the method could consistently detect the possible substitution of the Aureo with another variety, we investigated a more complex scenario of food fraud, in which the Aureo species is adulterated (mixed) with another variety, with different levels of contaminations. This is important in industrial settings to guarantee the mono-varietal nature of some productions. To this purpose, different mixtures of Aureo with decreasing contaminations of a G-type variant (from 50 to 10%) were prepared to probe the sensitivity of the strategy to also detect minor adulterations. As reported in After characterizing and optimizing the performance of our SNP test, we attempted to develop a simplified, instrument-free colorimetric assay with a naked-eye readout. Visual interpretation of the varietal discrimination was made possible by the reproducible and large time gap achieved between the fast and the slow reactions, in combination with pH-sensitive dyes that could provide a clear color change of the reaction tubes upon target amplification ,21. In pTo completely validate our system, a more complex case of adulteration was tested, in which the Aureo was mixed with two different G-type varieties of wheat . Remarkably, also in this case, the strategy revealed its high specificity, being able to correctly identify the different genomes in a complex mixture and even in the situation in which the total amount of the two contaminations was as low as 10% .In conclusion, we designed a single-SNP discriminating strategy to correctly identify mono-varietal productions based on Aureo, and we set up a direct colorimetric test able to discriminate, by simple visual inspection, the purity or the adulteration of Aureo with a wide range of G-type variants. The test exhibited high efficiency and specificity in complex cases of adulteration, in which different varieties were mixed, even at a 10% level of contamination. The isothermal nature of the LAMP amplification together with the naked-eye readout of the results make this approach potentially very useful in real quality control applications in the industry and food supply chain.