Early gastric cancer (EGC) is defined as a lesion restricted to the mucosa or submucosa, independent of size or evidence of regional lymph node metastases. Although computed tomography (CT) is the main technique for determining the stage of gastric cancer (GC), the accuracy of CT for determining tumor invasion of EGC was still unsatisfactory by radiologists. In this research, we attempted to construct an AI model to discriminate EGC in portal venous phase CT images.We retrospectively collected 658 GC patients from the first affiliated hospital of Nanchang university, and divided them into training and internal validation cohorts with a ratio of 8:2. As the external validation cohort, 93 GC patients were recruited from the second affiliated hospital of Soochow university. We developed several prediction models based on various convolutional neural networks, and compared their predictive performance.The deep learning model based on the ResNet101 neural network represented sufficient discrimination of EGC. In two validation cohorts, the areas under the curves (AUCs) for the receiver operating characteristic (ROC) curves were 0.993 (95% CI: 0.984-1.000) and 0.968 (95% CI: 0.935-1.000), respectively, and the accuracy was 0.946 and 0.914. Additionally, the deep learning model can also differentiate between mucosa and submucosa tumors of EGC.These results suggested that deep learning classifiers have the potential to be used as a screening tool for EGC, which is crucial in the individualized treatment of EGC patients. Gastric cancer (GC) is one of the most primary cancers and ranked as fifth and fourth in the global incidence rate and mortality rate in 2020 . The proFor the preoperative diagnosis of EGC, computed tomography (CT) and endoscopic ultrasonography (EUS) are the most commonly used methods. The main tumor can be seen on a CT scan, which can also measure the extent of tumor invasion and find nodal involvement and distant metastases . AccordiNowadays, Deep learning has been extensively discussed in the context of medical image analysis, including disease diagnosis, prognosis, and therapy. The deep learning-based models were trained utilizing enormous volumes of data from individuals with known messages, then the accomplished convolutional neural network (CNN) model may utilize data from other persons to predict their likelihood for that occurrence . Ole-JohThe cohort 1 in this study were 658 GC patients who had surgery in the first affiliated hospital of Nanchang University between June 2018 and December 2021. The training and internal validation cohorts were split up at random from the GC patients of cohort 1 with the ratio of 8:2. As the external validation cohort, 93 GC patients were recruited from the second affiliated hospital of Soochow university from January to December 2021. The following patients were excluded from the study , patientFor contrast-enhanced CT scanning, the following scanners were used: 128-channel CT , 256-channel CT (Philips Brilliance iCT 256), 256-channel CT  and 128-channel CT . The scanning specifications were as follows: a tube voltage range of 80 to 120 kVp, a tube current range of 120 to 300 mAs, a pitch range of 0.6 to 1.25 mm, an image matrix of 512×512, and a reconstruction slice thickness range of 1 or 2 mm. Before having an abdominal contrast-enhanced CT, each patient had an intramuscular injection of 20 mg of Racanisodamine Hydrochloride and drank 1,000-2,000 mL of water. Following intravenous injection of contrast media , the arterial phase and portal venous phase were recorded in 25-30 seconds and 65-70 seconds, respectively.th slice to be the one with the largest tumor lesion. Then, for further analysis, we isolated the (n - 2)th, (n - 1)th, nth, (n + 1)th, and (n + 2)th slices. These slices were saved as ‘png’ format. All GC pictures had standardized image contrast based on the abdominal window . The ROI was resized to 224×224 pixels, after being clipped out from these had extracted slices. During the portal venous phase, the tumor lesion was significantly increased and more easily separated from peripheral normal tissue, and many earlier studies employed this phase to segment tumor lesions , 22. Theth, (n - 1)th, nth, (n + 1)th, and (n + 2)th slices were extracted from one GC patients, the DL or DTL features of five slices were averaged to represent each GC patient. Furthermore, our research was implemented in Python 3.10 and run on a system with an Intel Xeon Silver 4214 CPU and 256 GB memory.In this study, we extracted DL and deep DTL features to constructed a prediction model, respectively. DL features were extracted based on various CNN models from every standardized ROI image, including ResNet18, ResNet34, ResNet50, ResNet101, ResNet152, Densenet121, Densenet201 and inception v3, which were pre-trained based on images of reality. For DTL features, the parameters of these CNN models were pre-trained by all of the ROI images from cohort 1. Then, the pre-trained CNN models were used to extract DTL features for each ROI image. The following process was utilized for all feature acquisition: ROI pictures were input into each CNN model or pre-trained CNN model, the average probability from all images was used to produce DL or DTL features, and the output from the penultimate FC layer was used as DL or DTL features Figure 2Each features group was employed individually to normalize combined features by z score normalization in the training, internal validation and external validation cohorts to merge features of various magnitudes into one magnitude. Non-zero coefficients served as useful predictors in each feature group using the absolute shrinkage and selection operator (LASSO) regression for feature selection in the training cohort . A statistically significant difference was determined to exist when P-value was less than 0.05. LASSO regression analysis and z score normalization were carried out using Python .Statistical analyses were performed with IBM SPSS Statistics  for windows. The Student’s P-value > 0.05), instead of gender. While, age, gender, tumor location, pathological T stage (T1 vs T2-4), lauren’s classification and CA19-9 level were shown significantly statistical differences between the training and external validation cohorts . Only tumor size, grade of differentiation, CEA level and CA12-5 level were not significantly different between the training and validation cohorts. This was mainly due to the limited sample size of the external validation cohort.As shown in We analyzed the performance of ResNet18, ResNet34, ResNet50, ResNet101, ResNet152, Densenet121, Densenet201 and inception v3 to determine the best model for diagnosing EGC Figure 3P-value of the DeLong test of different diagnosing EGC model in internal and external validation cohorts.In order to make the CNN model more suitable for medical diagnosis scenarios, we pre-trained the various CNN models and constructed diagnosing EGC models. For the pre-trained model, in two validation cohorts, the pre-trained ResNet101 also represented better diagnosis ability with AUC 0.965 (95% CI: 0.932-0.999) and 0.936 (95% CI: 0.886-0.986) than others Figure 4We used different machine learning classifiers to develop diagnosing EGC models, including SVM, KNN, decision trees, RF, extra trees, XGBoost and lightGBM. In all classifiers, the AUC value of SVM classifier represented better performance than other classifiers both in CNN and pre-trained CNN models 4. For eWith the advancement of human living conditions, the rise in the incidence of GC has drawn the attention of an increasing number of individuals. However, the majority of patients with GC received an advanced diagnosis after their initial diagnosis, which had a significant impact on their prognosis , 24. EGCTo our knowledge, various researches were kept on diagnosing T staging of GC, including CT, EUS, double contrast-enhanced ultrasonography and laparoscopic exploration , 26–28. The depth of invasion of EGC is an essential evidence in deciding on endoscopic resection . AbsolutIn order to choose the best CNN model for diagnosing EGC, we developed various diagnostic models based on different neural network models, including ResNet18, ResNet34, ResNet50, ResNet101, ResNet152, Densenet121, Densenet201 and Inception v3 in this study. We found that the performance of CNN models for diagnosing EGC was almost no significant difference using the DeLong test. It justly seemed the AUC value of the ResNet101 model represented the highest compared to others. Deep learning model training needs a significant amount of processing power . When chArtificial intelligence (AI) has advanced rapidly in biomedicine in recent years. The merging of AI technology and medical images such as endoscopy, radiographic images and pathology plays an important role in the diagnosis, staging and prognosis . Dong etThere are several limitations in this study . This stWe firstly constructed a CNN prediction model for diagnosing EGC from GC patients, and the deep learning model also had the potential for differentiating between mucosa and submucosa tumors of EGC. These results suggest CNN model can provide favorable information for individualized treatment of EGC patients.The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by The Ethics Committee of the First Affiliated Hospital of Nanchang University. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.QZ and ZF conceived the project and wrote the manuscript. YZhu and FZ drew the ROI of CT images. XS, AW, and LL participated in data analysis. YC and YT participated in the discussion and language editing. JX and ZL reviewed the manuscript. HL provided an external validation cohort. All authors contributed to the article and approved the submitted version.This work was supported by the National Natural Science Foundation of China (No.81860428), the leading scientists Project of Jiangxi Science and Technology Department (20213BCJL22050), and Youth Fund and Talent 555 Project of Jiangxi Provincial Science and Technology Department (20212BAB216036).We appreciate the Python technology offered by the OnekeyAI platform and thank the department of radiology for their support of CT images.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


People often worry about the side effects after vaccination, reducing the willingness to vaccinate. Thus, we tried to find out the risk of single nucleotide polymorphism (SNP) vaccines to improve the willingness and confidence in vaccination. Allergic and inflammatory reactions are the common vaccine side effects caused by immune system overreaction. In addition, a previous study showed significantly higher frequency of febrile reactions to measles vaccines in American Indians than in Caucasian children, indicating that the side effects varied in accordance with genetic polymorphisms in individuals. Thus, SNPs of immune regulatory genes, cytotoxic T-lymphocyte-associated protein 4 (CTLA4), CD28, tumor necrosis factor ligand superfamily member 4 (TNFSF4) and programmed cell death protein 1 (PDCD1) were included in this study to analyze their association with vaccine side effects. Moreover, 61 healthy participants were asked on the number of doses they received, the brand of the vaccine, and the side effects they suffered. We found that several SNPs were associated with side effects after the first or second dose of mRNA or adenoviral vector vaccines. Furthermore, these SNPs were associated with several autoimmune diseases and cancer types; thus, they played an important role in immune regulation. Moreover, rs3181096 and rs3181098 of CD28, rs733618 and rs3087243 of CTLA, and rs1234314 of TNFSF4 were associated with mild vaccine side effects induced by mRNA and adenoviral vector vaccines, which would play a potential role in vaccine-induced immune responses and may further lead to fatal side effects. These results could serve as a basis for investigating the mechanism of vaccine side effects. Furthermore, it was hoped that these results would address public concerns about the side effects of the COVID-19 vaccination. In clinical application, a rapid screening test can be performed to assess the risk of vaccine side effects before vaccination and provide immediate treatment. Recently, COVID-19 has caused a pandemic of severe acute respiratory syndrome (SARS-CoV-2). At present, the policies of various countries are inclined to coexist with the virus; thus, the vaccination rate must be improved. Although vaccination is a safe way of protecting people against infection and severe complications, people still worry about the side effects of vaccination. In addition, several studies have shown that the host genotype has a great effect on the immune response induced by vaccines, such as influenza, hepatitis B, and measles vaccination . TherefoVaccines are designed to stimulate antigen-specific T cell responses and enhance adaptive immunity. However, excessive T cell response may cause vaccine-induced side effects. Although the process of vaccine production and development have undergone rigorous testing, varying severities of side effects will be reported after large-scale vaccination. The most common side effects of COVID-19 vaccines are mild, such as soreness, swelling or redness at the injection site, fever, rash, and pain. Although the serious side effects are rare, they may cause seizures, thrombosis, myocarditis, or life-threatening allergic reactions . Fever iIn literature, no studies have investigated genotypes associated with the side effects of COVID-19 vaccines. However, SNPs susceptible to disease risk or disease severity may be involved in the immune response of vaccination, leading to vaccine-induced adverse reactions . TherefoIn this study, 61 healthy adults who received two doses of COVID-19 vaccine were enrolled from Chang Gung Memorial Hospital . Amongst them, 24 received two doses of mRNA-1273 (Moderna), 24 received two doses of ChAdOx1-S , 5 received two doses of BNT162b2 , 5 received MVC-COV1901 (Medigen), 2 received one dose of AZ followed by one dose of Moderna, and 1 received one dose of Moderna followed by one dose of BNT. Participants were asked on the number of doses they received, the brand of the vaccine and the side effects they experienced, including fever, pain, redness, or swelling at the injection site, shivering, nausea/anorexia, vomiting, diarrhoea, headache, fatigue, muscle pain, joint pain, and skin allergy. All data were self-reported. The study has been reviewed and approved by the Institutional Review Board of CGMH. The approved ID was 202101837B0. All the participants have signed the consent form.We collected oral mucosal cells of these participants to extract genomic DNA using the QIAamp DNA Mini Kit . Then, the purity and concentration of the extracted DNA were determined by measuring the absorbance at 260 and 280 nm. Then, the extracted DNA were stored for the following experiments.A total of four costimulatory genes  were investigated in this study. We focused on the promoter region of genes, because the SNP located in the promoter region may affect the expression level of gene . In addiThe Chi-square test and Fisher’s exact test were used to analyze the association between the vaccine-induced adverse reaction and the genotype frequency of participants using SPSS17.0. All data were analyzed through the dominant (AA vs. Aa+aa), recessive (AA+Aa vs. aa), and additive (AA vs. Aa vs. aa) models. Significant SNPs are summarized in A total of 61 participants who received COVID-19 vaccination were recruited in this study. All participants are Taiwanese. The complete characteristic data of side effects after vaccination in these cases are listed in p= 0.010; CAG/del +CAG vs. del, p=0.010). In addition, rs3181098 of CD28 was associated with discomfort at the injection site . rs733618 of CTLA4 , rs3181096 of CD28 , rs2227982  and rs36084323  of PDCD1 were associated with chills. Moreover, rs5839828 of PDCD1 was associated with diarrhoea based on the additive model (p=0.046). rs2227982  and rs36084323  of PDCD1 were associated with headache. Furthermore, rs1234314 of TNFSF4 , as well as rs2227982  and rs36084323  of PDCD1, was associated with myalgia. Two SNPs of CD28 were associated with skin allergy, namely, rs3181096  and rs200353921 .Analyzing the side effects induced by the first dose of mRNA vaccines Table 3,p=0.030; AG+AA vs. GG, p=0.013, OR=0.348, 95% CI. = 0.199–0.609) and rs36084323 . In addition, rs11571315  and rs231775  were associated with discomfort at the injection site. rs3181096 of CD28 , rs5839828 of PDCD1  and rs11571316 of CTLA4  were associated with chills. Moreover, rs3087243 of CTLA4 , rs3181097 of CD28 , and rs3181098 of CD28  were associated with headache. rs231775  of CTLA4 and rs3181097 of CD28  were associated with myalgia.Analyzing the side effects induced by second dose of mRNA vaccines Table 4,p=0.009; GG+GT vs. TT, p=0.009, OR=18.75, 95% CI. = 2.065–170.215). Three SNPs were associated with diarrhoea, namely, rs3181096 of CD28 , rs3181098 of CD28 , and rs1234314 of PDCD1 . Only the rs733618 of CTLA4 was associated with skin allergy .Analyzing the side effects induced by the first dose of AZ vaccines Table 5,p=0.027) and rs980967681 located in the 3’UTR . In addition, six SNPs were associated with diarrhoea, namely, rs1879877 , rs3181096 , and rs3181098  of CD28, rs3087243  and rs11571316  of CTLA4, and rs45454293 of TNFSF4 . Moreover, rs41386349 located in the promoter region of PDCD1 was associated with headache . rs733618 of CTLA4  and rs3181097 of CD28  were associated with myalgia.Analyzing the side effects induced by the second dose of AZ vaccines Table 6,Based on the results, rs3181096 and rs3181098 of CD28, rs733618 and rs3087243 of CTLA4 and rs1234314 of TNFSF4 were associated with side effects induced by mRNA and adenoviral vector vaccines. Furthermore, the data of mRNA and adenovirus vector vaccines were combined for analysis  or non-coding sequences (intron) of genes . If the In addition, rs3181096 of CD28, as well as rs2227982 and rs36084323 of PDCD1, was associated with side effects of mRNA vaccine in the first and second doses of vaccination. rs733618 of CTLA4, as well as rs1879877, rs3181096 and rs3181098 of CD28, was associated with the side effects of adenoviral vector vaccine in the first and second doses of vaccination. Considering that the components and principles are different in various vaccines, these SNPs may be related to the mechanism by which the components in the vaccine induce side effects. Moreover, these SNPs were associated with several autoimmune diseases and cancer types, which is summarized in The CD28 gene is located in the human chromosome 2q33 and the CD28 protein is primarily expressed on the naïve T cell. During T cell activation, CD28 plays an important role in providing a second signal for T cells when interacting with CD80/CD86 . Qi et aThe TNFSF4 gene is located in the human chromosome 1q25.1 and composed of 13 exons. It belongs to the TNF superfamily protein and it is the key to the coordination of innate or adaptive immune cells. This protein plays an important role in the life cycle of immune cells, such as differentiation, activation, inhibition, and apoptosis and it is involved in the pathogenesis of various autoimmune and inflammatory diseases  of Chang Gung Memorial Hospital (CGMH). The approved ID was 202101837B0. The patients/participants provided their written informed consent to participate in this study.D-PC: literature review and analysis, statistical analysis and interpretation of data, drafting of manuscript. Y-HW: critical revision of manuscript, literature review. F-PH/W-TL: performed the experiments, analysis and interpretation of data. All authors contributed to the article and approved the submitted version.This study was supported by grants to D-PC from Chang Gung Memorial Hospital (BMRPA81).We gratefully acknowledged Brian Tang and Enago (Crimson Interactive) for help as regards the poor English language of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Gracilariopsis lemaneiformis, the activities of four enzymes participating in the agar biosynthesis were detected, and phosphoglucomutase (PGM) was confirmed as highly correlated with agar accumulation. Three genes of PGM  were identified from the G. lemaneiformis genome. The subcellular localization analysis validated that GlPGM1 was located in the chloroplast and GlPGM3 was not significantly distributed in the organelles. Both the GlPGM1 and GlPGM3 protein levels showed a remarkable consistency with the agar variations, and GlPGM3 may participate in the carbon flux between (iso)floridoside, floridean starch and agar synthesis. After treatment with the PGM inhibitor, the agar and floridean starch contents and the activities of floridean starch synthase were significantly decreased; products identified in the Calvin cycle, the pentose phosphate pathway, the Embden-Meyerhof-Parnas pathway and the tricarboxylic acid cycle were depressed; however, lipids, phenolic acids and the intermediate metabolites, fructose-1,6-phosphate were upregulated. These findings reveal the essential role of PGM in regulating the carbon flux between agar and other carbohydrates in G. lemaneiformis, providing a guide for the artificial regulation of agar accumulation.Agar is widely applied across the food, pharmaceutical and biotechnology industries, owing to its various bioactive functions. To better understand the agar biosynthesis in commercial seaweed  Gracilaria/Gracilariopsis, which are the main genus of agar production. Among these agarophytes, Gracilariopsis lemaneiformis has a good agar yield of 10–30% and a gel strength of 140–1600 g·cm−2 [G. lemaneiformis has a superior yield and quality, there are still many problems to be solved, such as the complex agar extraction method, the high demands for raw materials and the incomplete agar accumulation pathway; all of these problems hinder the development of the agar industry.Agar, as a major important component of the extracellular matrix and the cell walls in some red algae, has great importance in the adaptation to marine environment. In addition, this sulfated polysaccharide possesses various bioactive functions, which are now widely applied across the food, pharmaceutical and biotechnology industries ,2. ApproGALT) and UDP-glucose pyrophosphorylase (UGPase) are positively correlated with the agar content of G. lemaneiformis [GME) in Gracilaria changii and Gracilaria salicornia exhibit different expression modes, corresponding to the different agar contents [G. lemaneiformis, the transcriptional expression level of PGM is proved to be highly related to agar biosynthesis [In the process of agar biosynthesis, two galactoses of β-D-galactose and 3,6-anhydro-L-galactose are first synthesized by a series of enzymes . Then, teiformis ,8. The tcontents . In G. lynthesis . HoweverAgar is situated at the end of carbon metabolism, does not decompose, but other metabolites, such as floridean starch and floridoside, can decompose into glucose-1-phosphate (G1P) and glucose-6-phosphate (G6P) that may be reused in the next round of synthesis of other carbon metabolites. Phosphoglucomutase (PGM), belonging to the phosphohexose mutase family, can regulate and control the convention of G1P and G6P, and be regarded as a major branch point of carbon metabolism in the formation of storage polysaccharides and energy production. In most higher plants, PGM plays a main role in the starch and sucrose synthesis . HoweverG. lemaneiformis has expanded rapidly along the coastal regions of China, which is mainly used for agar production, abalone bait and food industries. In addition, G. lemaneiformis has a high ability to remove and sequester carbon to attain carbon neutrality, which demonstrates the substantial ability of this seaweed in carbon use [GlPGMs, verified the enzyme’s subcellular location and expression modes, and analyzed the carbon flux among the diverse carbon storage materials under PGM-inhibited conditions in G. lemaneiformis. This study has an important guiding role in the artificial regulation of algal agar accumulation.In recent years, the important economic macroalga rbon use . In thisp < 0.05). In the nitrogen treatment, PMI, PGM and PMM showed the significant correlations with 0.896, 0.955, 0.888 on day 7, respectively. UGPase showed the highest positive correlation (0.864) with agar content on day 14; PGM revealed the positive correlation with agar content during the nitrogen treatment period. In the salinity treatment, only PGM showed a positive correlation at all of the time points. Combining with the results of the four enzyme activities and the agar content of G. lemaneiformis in different cultivation conditions, the PGM activities had the highest positive correlation , phosphomannomutase (PMM) and agar contents were simultaneously measured and compared in the different conditions . Of the relation . GlPGM1, GlPGM2 and GlPGM3 encode 585 aa, 580 aa and 572 aa, respectively. The molecular weights of GlPGM1, GlPGM2 and GlPGM3 are 63.14 kDa, 62.68 kDa and 61.97 kDa, with a theoretical isoelectric point (pI) of 5.02, 5.05 and 6.33, respectively. The conserved PGM_PMM_I domains with 141 aa in GlPGM1 and 146 aa in GlPGM2 are located at the N-terminal of the two GlPGMs. The conserved PGM_PMM_II domain is at the C-terminal of GlPGM3. The ORFs of A phylogenetic PGM tree and conserved motifs were constructed to show the evolutionary relationship between the related PGM sequences from large classes of plants . The treThe online prediction tools  agreed that GlPGM1 and GlPGM2 were located in the chloroplast. For GlPGM3, the CELLO Prediction showed that it was in the cytoplasm with 0.802 reliability, while WoLF PSORT revealed that it was located in the mitochondrion and Plant-mPLoc predicted it was in the chloroplast.G. lemaneiformis (G. lemaneiformis without incubation by antibodies (2) than in the cytoplasm (0.50 ± 0.06/μm2) (p < 0.05). However, the distributions of the colloidal gold particles of GlPGM3 were not significantly different between the cytoplasm, chloroplast or mitochondrion.Immunocolloidal gold transmission electron microscopy validated the locations of GlPGM1 and GlPGM3 in eiformis . To betttibodies . The chlG. lemaneiformis in Xiangshan (XS) were higher than that in other fields. The activities of GlPGM1 were the highest in the summer (May and July), and the lowest in the winter (January and March). The expressions of GlPGM3 in XS were significantly higher than that in other fields (p < 0.05). The GlPGM3 was significantly depressed in September and November  and the α-galactosidase (GLA) of (iso)floridoside were not significantly different from the above enzymes, the activities of the floridean starch synthase (SS) in the control were significantly higher than that in the inhibitor treatment at 24 h, and the activities of PMI were increased significantly at 3 h but repressed at 6 h .G. lemaneiformis in different treatments  was subsequently used for the multivariate statistical analysis. As shown in eatments B. The reTo further understand the role of PGM during the agar accumulation, the changes of the metabolites in the tricarboxylic acid cycle (TCA) cycle, the pentose phosphate pathway (PPP), the Embden–Meyerhof–Parnas pathway (EMP), the Calvin cycle (CC) and the agar pathways were compared . The PGMGracilaria; however, the results are contradictory, partly because of the different methods used for agar extraction, and the nature of seasonal changes varies geographically. The lowest and highest yields of Gracilaria veleroae agar occurred in the summer and autumn, respectively, while the highest agar yields for Gracilaria vermiculophylla occurred in the winter [G. lemaneiformis was found in summer (May and July in 2020) and a richer agar content occurred in Xiangshan. The yield of agar can be altered with the changing environment. Low salinity, nitrogen deficiency and phosphate enrichment can significantly promote the agar contents ,15. Heree winter . A signiG. lemaneiformis, the expression of PGM is closely related with the agar content after application of 24-epibrassinolide [UGPase could also be found at different life stages and in different cultivation fields [G. lemaneiformis. These variations of relevance may be because other materials share the same upstream of the pathway and agar is at the end of metabolism.The enzymes in agar synthesis can modulate the content of substrates and thus affect the agar content. The four enzymes measured in this study showed positive correlations with agar variations under different treatments, and PGM exhibited the significant correlation with agar content. PGM may regulate the carbon flux between the different materials in marine algae. In sinolide , which in fields ,17, whicPGM genes are screened in Arabidopsis thaliana, Oryza sativa and Zea mays. The numbers of PGM genes in seaweeds are relatively less than those in higher plants. Four PGM sequences are found in red algae, such as Gracilariopsis chorda, Chondrus crispus and Porphyra umbilicalis. However, no PGM gene is found in brown seaweed, while the bifunctional enzyme PMM can function as PMM and PGM [PGM sequences were obtained through screening the genome database of G. lemaneiformis. In this study, the whole evolutionary tree of the PGM family is divided into three clades, because of the conserved motif. There is a close sequence similarity between these PGMs in different species and they are descended from ancestral gene via gene duplication and speciation events. GlPGM1 and GlPGM2 have a similar conserved motif and are clustered into the clade I, from which it can be concluded that the homologous PGMs are similar in the biological role. GlPGM3 is close to red algae, followed by higher plants, which indicates that these PGM sequences from higher land plants could be evolved from red algae. Meanwhile, the tree implies that gene duplication can give rise to the production of in-paralogs in a species and also out-paralogs in different species [With the development of high throughput sequencing and bioinformatics technology, a large number of gene families have been excavated. Numerous  and PGM . A total species . Arabidopsis [Zea mays. The plastidial PGM in potatoes forms part of the starch in the generated transgenic potato plants [Two isoforms, plastidial and cytosolic PGMs, display high activities in potato tubers, bidopsis , tobaccobidopsis  and Zea o plants . The plao plants . Howevero plants ,24. Howeo plants . The rolo plants , (iso)flo plants . The incTo better explore the role of PGM in the pathways of agar biosynthesis and carbon flux, different inhibitors of PGM were chosen. Some of the endogenous intermediates and related structural analogs, such as adenosine triphosphate (ATP), citrate , fructosG. lemaneiformis. In addition, the activities of PMI varied due to the PGM inhibition. However, other enzymic activities and related metabolites were not detected, so the function of PGM on 3,6-anhydro-L-galactose synthesis should be further analyzed. SS is a key regulatory enzyme involved in the starch biosynthesis and is differently regulated in different tissues . The actG. lemaneiformis. Earlier studies have shown that carbohydrates and the lipid biosynthetic pathways compete for the substrates, as the biosynthetic pathways utilize the same precursors. PGM overexpression can accumulate the production of chrysolaminarin, while the lipid contents are reduced [G. lemaneiformis, (iso)floridoside, starch and agar accumulations are the main carbon sinks in the cells. It is also important to know how carbon flow is regulated by the carbohydrate and lipid metabolism in Rhodophyta, which is important for biofuel manipulations [In the present study, the inhibition of PGM resulted in the overall increase in the lipid contents while a decrease in carbohydrates, such as (iso)floridoside, floridean starch and agar, which implied a possible carbon flux between the lipids and the energy storage materials in  reduced . The car reduced . In G. lulations . For the carbon flux under PGM inhibited condition, the Cavin cycle, the EMP pathway, the TCA cycle and agar biosynthesis showed a downward trend, whereas fructose-1,6-phosphate exhibited a significant increase. During the biological pathway, fructose-1, 6-phosphate is an important intermediate in EMP and PPP. It can be cleaved into two trioses, dihydroxyacetone phosphate and glyceraldehyde-3-phosphate, which are the substrates of carbohydrates and lipid biosynthesis . In the G. lemaneiformis 981 was collected from the coast of Xiapu , Fujian Province, China. In the laboratory, the thalli were cultivated in the transparent aquaria at an irradiance of 30 µmol photons m−2·s−1 and a 12 h: 12 h light: dark cycle at 23 °C for one week. The tank contained 5 L filtered natural seawater  filled with filtered air and the seawater was changed every day. These algal materials were used for correlation analysis, inhibition treatment and Western blotting analysis. The alga of The salinity and nitrogen experiments for the agar and enzyme correlation analysis were carried out in the laboratory. In each culture treatment, approximately 15 g of fresh thalli were placed into 10 L seawater and each treatment took three replicates. For the salinity treatment, the thalli were placed at a salinity of 17 and 33 with normal Provasoli medium, respectively. The samples for agar and enzymic analyses were collected on 0 day, 7 day, 14 day, 21 day, respectively. For the nitrogen treatment, the thalli were cultured in seawater  with normal (HN) and nitrogen-limited Provasoli medium (LN), respectively. The samples for the agar and enzymic analyses were collected on 0 day, 7 day, 14 day and 21 day, respectively. Apart for the nitrogen and salinity factors, the other culture conditions were consistent with the adaptation culture condition.G. lemaneiformis 981 was collected from Xiapu in January, March, May, July, September, November of 2020. In the different cultivation fields’ experiment, the algae were collected from Xiapu, Lianjiang in Fujian Province, Xiangshan in Zhejiang Province in July of 2020, respectively. Intact and healthy thalli were selected from floating lines and placed in liquid nitrogen for analysis.In the cultivation months experiment, the alga of G. lemaneiformis was determined, using the alkali treatment method [Approximately 5 g of fresh thalli from the aforementioned treatments were used to assay the agar content. The agar content of t method . AccordiThe activities of the enzymes involved in the agar biosynthetic pathway were analyzed. Of them, PMI activity was detected according to Maruta et al. ; PGM andG. lemaneiformis were captured from our own laboratory database [http://pfam.xfam.org/, 29 June 2022) were used as the query to search the G. lemaneiformis protein database with an e-value ≤ e−10. Blastp was subsequently used to search for missing possible PGM candidates. Pfam and SMART  were then used to confirm the conserved domain, and the protein sequences lacking the conserved domains were excluded. The whole genome sequences of database . For catG. lemaneiformis was isolated by a RNeasy Plant mini kit , according to the manufacturer’s protocol. The cDNA for the full-length sequence cloning was subsequently synthetized by using HiScript II Reverse Transcriptase Kit . The opening reading frames (ORFs) of three possible GlPGM were annotated as GlPGM1, GlPGM2 and GlPGM3, and finally amplified by PCR, using Prime Star Kit . The gene-specific primers with different restriction endonuclease site are indicated in the additional file The total RNA from s of GlPGM1, GlPGM2 and GlPGM3 were calculated by the ProtParam . The conserved structural domains were constructed in the NCBI Conserved Domain Search. The protein sequences annotated as PGM from archaea, cyanobacteria, fungi, algae, and terrestrial plants were retrieved and collected from NCBI, JGI , TAIR  and the Ensembl  database, respectively. The PGMs of these species were presented on the additional file https://meme-suite.org/meme/, 29 June 2022) was used for searching the motifs of these PGM sequences. The phylogenetic tree was subsequently constructed, using maximum likelihood methods with 1000 bootstrap replicates. The theoretical molecular weights and pIGlPGM1 and GlPGM3 were amplified, as mentioned above, and then cloned into the pMD18-T vector  for sequencing . The GlPGM1 and GlPGM3 from the pMD18-T vector were then cut, using restriction endonuclease and cloned into expression vector pET-28a (+)  to obtain pET-28a-GlPGM1 and pET-28a-GlPGM3 recombinant strains, respectively. The constructed plasmids were transformed into the expression strain E. coli BL21 . The recombinant strains of GlPGM1and GlPGM3 were incubated and induced with 1mM isopropyl-β-D-thiogalactopyranoside (IPTG) for 3–5 h at 28 °C. The overexpressed proteins GlPGM1and GlPGM3 were purified and checked by 12.5% SDS-PAGE. The E. coli with recombinant pET-28a without IPTG was used as the negative control. The ORFs of G. lemaneiformis . The purified proteins, GlPGM1 and GlPGM3, were used as antigens to immunize rabbits for the production of the polyclonal antiserum. A Western blot analysis with the antibodies was conducted to check the expression of GlPGM1 and GlPGM3 from eiformis .http://cello.life.nctu.edu.tw/, 29 June 2022), WoLF PSORT tool  and Plant-mPLoc . First, the online prediction tools were used to predict the possible subcellular localizations of GlPGM1, GlPGM2 and GlPGM3, using Cello v2.5  overnight and then dehydrated, using a gradient of ethanol concentrations in −20 °C. The dehydrated thalli were infiltrated with a gradient of LG Gold ethanol concentrations and embedded in 0.1% BENZIL in LG Gold ethanol for a week, followed by sectioning with a LEICA EM UC7 ultramicrotome instrument . The sections were blocked for 2 h at room temperature and then washed three times with TBST, followed by incubating with polyclonal antibody against GlPGM1 and GlPGM3, respectively. The sections were finally incubated with goat anti-rabbit IgG H&L (15 nm Gold) 1 h and examined with a TEM instrument . Immunoblotting analysis was performed, as described in . The totThe enzyme activities participated in agar biosynthetic pathway and other carbon metabolisms were analyzed at 0 h, 3 h, 12 h, 24 h, respectively. The activities of the enzymes  were measured, using the similar methods described previously. The activities of possible (iso)floridoside synthase TPS and degrading enzyme GLA were detected, according to the previous descriptions ,41, respAn Ultra Performance Liquid Chromatography-electrospray ionization-tandem mass spectrometry (UPLC-ESI-MS/MS) system was used to analyze the metabolites. The extraction, detection, qualification and quantification of the metabolites were performed at Metware Biotechnology Co., Ltd. , following the method described previously . The flop > 0.05) and Levene’s test (p > 0.05), respectively. The effects of the treatments, the time, and their interactions were assessed by a two-way analysis of variance (ANOVA). A Tukey post hoc test (Tukey HSD) was performed to show the differences between the treatments and time. The significance level was set at p < 0.05.The correlation between the enzyme activities and agar contents was conducted by Pearson correlation analysis . The normal distribution of all of the data under each treatment and the homogeneity of variance were confirmed by a Shapiro–Wilk test (G. lemaneiformis, and explored the function of PGM in carbon metabolic pathways, especially in agar biosynthesis (This study revealed the high correlation between PGM activities and agar contents in ynthesis . GlPGM1


Rac gene family participates in diverse signal transduction events, cytoskeleton morphogenesis, reactive oxygen species (ROS) production and hormone responses in plants as molecular switches. S. album is a popular semiparasitic plant that absorbs nutrients from the host plant through the haustoria to meet its own growth and development needs. Because the whole plant has a high use value, due to the high production value of its perfume oils, it is known as the “tree of gold”. Based on the full-length transcriptome data of S. album, nine Rac gene members were named SaRac1-9, and we analyzed their physicochemical properties. Evolutionary analysis showed that SaRac1-7, AtRac1-6, AtRac9 and AtRac11 and OsRac5, OsRacB and OsRacD belong to the typical plant type I Rac/Rop protein, while SaRac8-9, AtRac7, AtRac8, AtRac10 and OsRac1-4 belong to the type II Rac/ROP protein. Tissue-specific expression analysis showed that nine genes were expressed in roots, stems, leaves and haustoria, and SaRac7/8/9 expression in stems, haustoria and roots was significantly higher than that in leaves. The expression levels of SaRac1, SaRac4 and SaRac6 in stems were very low, and the expression levels of SaRac2 and SaRac5 in roots and SaRac2/3/7 in haustoria were very high, which indicated that these genes were closely related to the formation of S. album haustoria. To further analyze the function of SaRac, nine Rac genes in sandalwood were subjected to drought stress and hormone treatments. These results establish a preliminary foundation for the regulation of growth and development in S. album by SaRac.Plant-specific Rac/Rop small GTPases, also known as Rop, belong to the Rho subfamily. Rac proteins can be divided into two types according to their C-terminal motifs: Type I Rac proteins have a typical CaaL motif at the C-terminal, whereas type II Rac proteins lack this motif but retain a cysteine-containing element for membrane anchoring. The  S. album is a small semiparasitic tree belonging to the genus Santalum in the family Santalaceae. This family is composed of 29 genera with approximately 400 species, 19 of which are specific to the Santalum genus [S. album is known as the “tree of gold”, and its heartwood is widely used in high-end craft sculpture and high-end furniture manufacturing [S. album grows for a long time; generally, it begins to form heartwood after 7 or 8 years of growth in natural environments. Unsustainable exploitation of wild trees, combined with increasing global demand for sandalwood products has threatened native sandalwood populations in some places, such as southern India. Because of the contradiction between the increasing market demands for its heartwood and the shortage of S. album heartwood on the market, the shortage of heartwood with its destruction of natural resources has become increasingly complicated [um genus . It is dacturing ,6. The eacturing ,8. Sandaacturing . S. albuplicated .S. album is a semiparasitic plant; its roots have a special organ, the haustorium, which, through the haustorium’s contact with the host plant root, absorbs its own water and nutrients [S. album and the formation of heartwood. As far as the current scientific progress is concerned, the study of the regulatory genes related to haustoria in S. album is still limited.utrients . TherefoROS can regulate plant growth and development, hormone transduction, root hair development and so on. In sandalwood, Rboh is significantly induced by the haustorial inducer DMBQ, and the ROS signal produced by Rboh protein is necessary for the development of sandalwood haustoria . At presRac gene family have been shown to regulate the growth and development of S. album through ROS production. Based on previous studies, nine Rac genes were obtained from the transcriptome and genomic data of S. album and compared with eleven Rac family members in A. thaliana and seven Rac family members in rice. The bioinformatics data of these sequences and tissue-specific expression were analyzed and these results will provide a basis for further analysis of the related functions of S. album Rac genes and its relationship with haustorium formation. Additionally, the expression levels of SaRac1-9 under drought stress and hormone treatments were studied, which play an important role in the growth and development of sandalwood.Plant-specific Rac small GTPases belong to the plant Rho subfamily . In animThe materials used in the experiment are stored in this laboratory, Institute of Tropical Forestry, Chinese Academy of Forestry Sciences . Four tissues were collected: root, stem, leaf and primary haustorium , each with three biological replicates, and stored in a −80 °C refrigerator. Sandalwood seedlings grew in a greenhouse in the soil for approximately 2 months, until they reached 15 cm. Saplings displaying similar growth were subsequently used for analysis. The soil water was withheld to drought for 0, 3 and 9 d, in which the soil relative water content was reduced from 70% to 32%. The soil relative water content of control plants was maintained at 80%, which represents a suitable soil environment for sandalwood growth. For hormone treatments , the plants were divided into four zones , which were treated with different hormones at a concentration of 100μM/L. Three biological replicates were included. After 48 h, samples were collected at the same time and immediately immersed in liquid nitrogen.Rac was obtained, and the DNA sequence and promoter sequence of Rac were obtained from S. album genome data by homologous alignment. The nucleotide sequence homology of Rac in sandalwood was analyzed using TBtools.According to the gene annotation information of the sandalwood transcriptome, the coding sequence of https://meme-suite.org/meme/, accessed on 13 July 2022). By selecting a motif of full length, eight conserved motifs located in the SaRac domain were identified. To analyze the cis-regulatory element in the putative SaRac promoter, we performed a cis-regulatory element analysis of the 2000 BP promoter sequence of the nine SaRac genes using TBtools and the Plant CARE website , and their functions and numbers were predicted.The conserved domains of nine SaRac proteins were predicted using NCBI-Conserved Domain Search, and the structure of SaRac was analyzed using TBtools. Novel putative motifs were explored using the MEME server . Protein subcellular localization was predicted by using the WoLF PSORT website .The basic physical and chemical properties of the protein, including molecular weight (MW), isoelectric point (pI) hydrophilicity and number of amino acids, were analyzed using ProtParam online software , and the 7 Rac members of rice were obtained from NCBI  using the MUSCLE method. A phylogenetic tree was constructed by using the NJ method implemented in MEGA-X. The parameters for tree construction were as follows. Phylogenetic analysis parameters: bootstrap (1000 replicates); gaps/missing data: pairwise deletion; model: Dayhoff model; pattern among lineages: same (homogeneous) and rates among sites: uniform rates. Finally, the phylogenetic tree was constructed.A multiple sequence alignment of 27 Rac proteins from SaRac genes on chromosomes of S. album were obtained from the gene annotation information of the S. album transcriptome. The chromosomal locations were displayed with TBtools . The numbers and organization of introns, exons and gene structures were drawn and displayed using TBtools.Positional information and gene structures of SaRac genes were obtained by TBtools software. Using TBtools software, the synteny relationships of orthologous Rac genes among S. album, A. thaliana and rice were evaluated. The parameters for collinearity of the SaRac gene were as follows: the genome file was used to construct the chromosome skeleton to obtain the gene density file, and then TBtools was used to prepare the collinearity file, extract the location of the SaRac gene, highlight the collinearity region of the target gene and finally, visualize it.The chromosomal locations of S. album. The SaRac1-9 gene sequence was obtained by reverse transcription of cDNA with a reverse transcription kit , and quantitative primers were designed according to the whole-field sequence of the SaRac1-9 gene . The qPCR mixture were 0.5 μL of primers, 1 μL of cDNA, 10 μL of 2 × Mix and 8 μL of dd H2O. The reaction procedure was 95 °C for 10 s, 60 °C for 10 s and 72 °C for 20 s, and 40 cycles were performed. Three biological replicates and 3 technical replicates were carried out. SPSS 25 software was used to analyze the significance of the data, and OriginPro 2019b software was used to draw the graph.According to the instructions of the plant RNA extraction kit , total RNA was extracted from 1-9 gene . Real-tiSaRac were isolated from the transcriptome and genome of S. album and named SaRac1-9. The basic physical and chemical properties of the protein were analyzed by ProtParam  (To clarify the distribution of  members A. The Sa/SaRac1) A,B (Tabl/SaRac1) .A. thaliana and rice, we constructed two syntenic maps of sandalwood with A. thaliana and rice. A total of three SaRac genes showed syntenic relationships with Racs in O. sativa  and GTP binding sites . The G2 and G3 domains are also called switch I and II loops, and the C21 and C156 positions are the conserved L-cysteine residues of the G domain , MgG domain .Rac gene family members in roots, stems, leaves and primary haustoria of S. album by green fluorescent quantitative PCR with gene-specific primers. Nine genes (SaRac1-9) were expressed in roots, stems, leaves and primary haustoria, but there was a difference in their expression levels. The expression of SaRac7/8/9 in stems, haustoria and roots was higher than that in leaves, but the expression of SaRac1, SaRac4 and SaRac6 in stems was lower in leaves. The expression levels of SaRac2 in stems and primary haustoria were high. It is worth noting that the expression levels of SaRac2 and SaRac5 in roots were approximately 6 and 13, respectively, relative to leaves  production and phytohormone response, and play a very important role in the abovementioned events ,46. Rac Reactive oxygen species (ROS) produced by NADPH oxidase have been shown to play many important roles in signaling and development in plants, such as in plant defense response, cell death, abiotic stress, stomatal closure, and root hair development ,51,52,53Rac genes with drought and hormones. Only two genes, SaRac6 and SaRac8, showed higher expression under long-term drought stress than those under control conditions. Auxin, abscisic acid (ABA) and ethephon play key roles in the development of many plants. These three hormones are often used as the main substances in plant hormone response experiments. In this study, we treated nine SaRac1, SaRac3, SaRac7 and SaRac8 were higher under ABA treatments. Moreover, more than half of the genes, including SaRac1, SaRac3, SaRac5 and SaRac6, showed higher expression under ethephon treatments. Furthermore, the expression levels of SaRac1, SaRac6, SaRac7 and SaRac8 increased after 48 h of IAA treatments, indicating these genes are responsive to IAA. Previous studies had demonstrated that auxin biosynthesis is essential for haustorium in haustorium formation in the root-parasitic plants [S. album haustorium and further influence the growth and development of sandalwood.The results of hormone treatments indicated that the expression of c plants ,56. ThusRac gene family in sandalwood, the structure, conserved motifs, phylogenetic relationships and collinearity of SaRac genes were characterized. One conserved motif was located in the Rac domain, suggesting that the Rac domain is conserved among A. thaliana, rice and sandalwood. Most of the SaRac genes exhibited similar numbers of exons. Phylogenetic analysis revealed that SaRac, AtRac and OsRac proteins could be classified into five subgroups. Groups IV and V contain more Racs that come from all three species, and groups I and II also contain Racs from all three species. Moreover, group III contains one Rac in A. thaliana. For instance, SaRac5 to SaRac9 in group IV and SaRac4 to AtRac1 in group V could have expansive functions surrounded by AtRac, SaRac or OsRac proteins with different functions.In our study, to better understand the evolution of the A. thaliana, and three pairs were detected in rice, indicating a strong homologous relationship between sandalwood and A. thaliana, and a weak homologous relationship between sandalwood and rice.Additionally, synteny maps between two representative species and sandalwood were constructed to better understand the phylogenetic relationships. More than 10 pairs were detected in SaRac genes that were assigned to segmental duplication events, implying high segmental duplication. These results indicated that some SaRac genes were possibly generated by gene duplication.Gene duplication is a major mechanism underlying the evolution of novel protein functions. We detected five A. thaliana and rice, and their roles include the regulation of root hair initiation and apical growth, hormonal responses, stress responses and so on. Among them, the best-described Rac proteins are the members of group IV  was found in the promoters of SaRac2-9, and the gibberellin response element (P-box) was found in the promoters of SaRac1/2/4/5/6/7/8/9 However, SaRac2/3/5/8/9 all had TC-rich repeats related to defense and stress responses, suggesting that these Rac genes may be involved in disease resistance and stress resistance of S. album.In gene expression, promoters play an important role in regulation, through which gene expression can be changed to change the characteristics of plants. Therefore, the study of promoters is a key step in genetic engineering and gene expression research . In addiSaRac members more accurately, based on the functional prediction of gene promoters, in this study, the expression levels of nine genes at four sites were detected by green fluorescent quantitative PCR. The results showed that, compared with the expression levels of leaves in each gene, the expression levels of SaRac1, SaRac4 and SaRac6 in stems were very low, while the expression levels of SaRac2 in stems and primary haustoria were very high, and the expression levels of SaRac2 and SaRac5 in roots were approximately 6 and 13, respectively. In general, the level of protein expression in specific tissues is closely related to its function. The high expression of SaRac2 and SaRac5 in roots and SaRac2/3/7 in haustoria may indicate that these genes are closely related to the formation of haustoria in S. album.To understand the function of nine S. album at present, in this study, the biological information and expression pattern of 9 Rac genes in S. album were analyzed (Rac family genes involved in the growth and development of S. album and regulation its functions. The perspectives of research on the semiparasitic sandalwood will develop towards the Rac-dependent generation of ROS in promoting haustorium development, which more effectively provides a data base for the growth mechanism of sandalwood.Because of the lack of research on the molecular mechanism of growth and development in analyzed . These r


Glucose-galactose malabsorption is a rare inherited autosomal recessive genetic defect. A mutation in the glucose sodium-dependent transporter-1 gene will alter the transportation and absorption of glucose and galactose in the intestine. The defect in the SGLT-1 leads to unabsorbed galactose, glucose, and sodium, which stay in the intestine, leading to dehydration and hyperosmotic diarrhea. Often, glucose-galactose malabsorption patients are highly dependent on fructose, their primary source of carbohydrates. This study aims to investigate all published studies on congenital glucose-galactose malabsorption and fructose malabsorption. One hundred published studies were assessed for eligibility in this study, and thirteen studies were identified and reviewed. Studies showed that high fructose consumption has many health effects and could generate life-threatening complications. None of the published studies included in this review discussed or specified the side effects of fructose consumption as a primary source of carbohydrates in congenital glucose-galactose malabsorption patients. Carbohydrates are one of the three core nutrients the human body needs in our daily diet, besides fat and protein. Carbohydrates are a cover term that contains essential food groups such as fruits and vegetables, dairy products, fibers, and legumes. It makes up about 45% to 65% of our calorie intake ,2,3. Car6H12O6) is the simplest carbohydrate in the world, and all living organisms are highly dependent on this monosaccharide. Glucose is the human body’s essential energy source in aerobic and anaerobic cellular respiration [The psychological level of the patient and the patient’s family may be affected due to the patient’s altered lifestyle, the fear of further negative progression of the condition, and frequent visits to healthcare facilities. In some cases, the patient may require staying for long periods admitted to the hospital, which will be stressful for both the patient and the family. Glucose C6H2O6 is thpiration . It is tGlucose transporters have many differences like tissue type, affinity, and capacity, but the central aspect of difference is the reliability of sodium. There are two main types of glucose transporters. The first one is sodium-dependent glucose transporter-1 (SGLT-1), using the active transport method, and the second type is sodium-independent glucose transporter (GLUT) which uses facilitated diffusion . FructosGalactose is a monosaccharide found in dairy products in the form of lactose. It is a disaccharide formed of a condensation reaction between glucose and galactose, linking them together to form lactose; galactose is transported into the enterocyte by SGLT-1 and transported out to the bloodstream by GLUT2 . One of GGM is a rare genetic disorder affecting a few of our populations and is responsible for some deaths in newborns in early life. Most cases are Arabs, especially in Saudi Arabia . CarbohyCongenital glucose-galactose malabsorption (CGGM) is a rare autosomal recessive disorder driven by a galactose and glucose transportation defect across the small intestine . CGGM isThe defect in the SGLT-1 leads to unabsorbed galactose, glucose, and sodium, which stay in the intestine, leading to dehydration and hyperosmotic diarrhea ,30. ThusFurther complications might occur as well, such as kidney injury, nephrolithiasis, and hypercalcemia ,33. LuckA literature review of published studies in PubMed, Google Scholar, ScienceDirect, Saudi Digital Library and the King Saud University library was conducted from inception up to October 10, 2022, using an algorithm based on relative keywords such as “glucose-galactose malabsorption”, “fructose consumption”, “metabolic syndrome”, “carbohydrates malabsorption”, “diarrhea”, “SLC5A1”, “SGLT1”, and “autosomal recessive genetic defects”.Inclusion criteria include studies that examine the molecular basis and diagnostic methods of CGGM. Also, clinical and personal characteristics of confirmed cases of CGGM, including diagnosis, complications, treatment, country of birth, and nationality of the cases diagnosed. Although, the onset of diarrhea after birth, evidence of carbohydrate malabsorption with a positive test, reducing substance in the stools, and failure to improve lactose-free and amino acid-based elemental formula given in sequence. In addition, a remarkable improvement in diarrhea after the elimination of glucose and galactose using the fructose-based formula; recurrence of diarrhea following exposure to glucose or lactose. Also, articles that examined fructose consumption and its complications were included in this study. Exclusion criteria include chronic diarrhea and non-English studies. Few published studies have been conducted partially on related interest research. As shown in Over the last decades, clinical research and epidemiological studies have aimed to report the laboratory and clinical characteristics of patients with CGGM and reduce malnutrition risk factors in the targeted populations. Several factors have been reported addressing the effect of the complications of CGGM.In a literature review that included 107 CGGM cases, 47 (43.9%) were from Saudi Arabia, 34 (31.8%) were from the United States of America, 9 (8.4%) were from Turkey, 3 (2.7%) were from China, 2 (1.9%) were from the United Kingdom, 2 (1.9%) Canada, and 2 (1.9%) Japan. One case for each was reported from Jordan, Morocco, India, Egypt, Belgium, Macedonia, Iran, and Oman (0.9% for each). Dehydration was stated in all cases, diarrhea was observed in 104 cases , vomiting was reported in 11 cases , inadequate weight gain was occurred in 86 cases , abdominal distension was stated in 51 cases , fever was occurred in 3 cases , hematuria/polyuria was observed in 7 cases , and hypernatremia was stated in 57 cases . The CGGM complications include kidney injury, hypercalcemia, and nephrolithiasis, which were observed in 20 cases  . In someThe SGLT-1 defect results in unabsorbed galactose, glucose, and sodium, which remain in the intestine, causing dehydration and diarrhea. In humans, several factors regulate the transcription of SGLT1 at the promotor region. For example, the binding of hepatic nuclear factor (HNF) 1, transcription factor SP1, and cAMP response element–binding protein (CREB) to the 5′ region of the SGLT1 gene and their impacts on transcription have been documented ,44.The studies of the isolated pedigree of GGM patients confirmed the autosomal recessive mode of inheritance. Thirteen GGM cases were added to the pedigree and traced back to the late 17th century. They found that the insertion of the Q457R) protein into the plasma membrane is impressive as other mutations lead to trafficking defects between the endoplasmic reticulum and the membrane among GGM cases. The failure to translocate bound glucose across the membrane is related to the glucose transport defect by the (Q457R) ,14. The 7R protein = 3), bloating (n = 3), and abdominal discomfort (n = 1). Few children from this cohort persisted in having symptoms of sugar malabsorption during the follow-up. All the subjects had normal development, and none of them had neurological disorders or complications. It was well reported that subjects with CGGM develop symptoms even in maturity with little glucose exposure. The hydrogen breath test stays positive despite thriving well on the fructose replacement formula [A retrospective study was conducted in the western Province of the Kingdom of Saudi Arabia. The study included 24 CGGM patients with a median age of 4.5 months. Half of the subjects were males, 66.7% were Saudi 33.3% were non-Saudi (3 Asians and 5 Arabs). They amid to report the clinical and laboratory characteristics of CGGM patients. They found that 37.5% had affected siblings with CGGM, and 87.5% of the parents were consanguineous. The common symptom was diarrhea and dehydration. Renal stones and nephrocalcinosis were reported in 3 (12.5%) CGGM patients at eight months, twelve months, and seven years, respectively. Renal tubular acidosis and hypernatremia were reported in 16.6% and 29.2%, respectively. During the follow-up, the median follow-up duration was 41.6 months. All except three revealed normal weight gain. Five patients reported one or more symptoms of diarrhea  among participants who were healthy adults aged 18–60 years, with a BMI ranging from 19 to 39 kg/mng FGF21 ,47. Moreng FGF21 ,48,49. Ing FGF21 ,51,52,53ng FGF21 .A published study focusing on leptin resistance found that leptin resistance developed silently without detecting biochemical or physiological differences between the fructose and control groups and that a high fructose diet promotes obesity. Also, the effects of chronic fructose consumption can develop silently without any metabolic syndrome and visible signs of elevated leptin levels . When frThere are some consequences to high fructose consumption, known as fructose malabsorption. Fructose malabsorption is caused by an overload of the intestinal transport capacity for fructose, distinguishing it from the rare hereditary fructose intolerance  ,59. The Despite the considerable heterogeneity of published literature, including several further limitations reported herein, CGGM plays a crucial role in the malnutrition status and clinical outcomes, therefore conducting necessary studies and analyzing its relation to fructose malabsorption management would facilitate a proper nutritional intervention targeting the best nutrition, and typical development of CGGM patients.Although there are health threats to the high consumption of fructose, a few subjects in our populations are obliged to depend on fructose as their primary source of carbohydrates in their diet. These individuals are CGGM patients, and their suffering from this metabolic disorder is harsh, and its complications threaten their lives. Understanding the metabolism of fructose and the relationship between excessive consumption of fructose and metabolic disturbances provides another opportunity to prevent and treat many chronic diseases. Future studies with adequate power and longitudinal design are nonetheless required to assess further the association of nutritional status with CGGM clinical outcomes using a more standardized definition to measure nutritional status in this population.


The purpose of this study was to evaluate the safety, tolerability, and pharmacokinetics (PKs) of rhEGF eyedrops after the administration of single and multiple doses in healthy subjects. A phase 1, randomized, double-blind, placebo-controlled, and single-ascending dose (SAD) and multiple-ascending dose (MAD) study were conducted in three dose groups . The subjects randomly received rhEGF eyedrops or the placebo in a 3:1 ratio. Serial blood and tear samples for PK analysis were collected up to 36 h and 180 h post-dose in SAD and MAD studies, respectively. In addition, the serum and tear EGF concentrations were measured. Immunogenicity evaluations were conducted using serum anti-EGF antibody levels. A total of 50 subjects were enrolled and 48 subjects completed the study. Adverse drug reactions were mild and transient. There were no serious adverse events in this study. The tear EGF concentrations rapidly increased and returned to baseline after 4 h without any serum EGF level change after the administration of rhEGF eyedrops. rhEGF eyedrops were safe and well-tolerated in healthy subjects in a dose range of 10–100 μg/mL, indicating suitability for further studies in patients with corneal injury. Corneal injury is a common disease, accounting for approximately 3% of emergency department visits . The corCorneal healing is a complex process involving cell death, migration, proliferation, differentiation, and extracellular matrix remodeling . In addiEpidermal growth factor (EGF), one of the growth factors that are secreted from the lacrimal gland , is invoFor the treatment of corneal injury, the topical eye administration of EGF has been used through cord blood serum, autologous serum, amniotic membrane extract, and amniotic membrane transplantation ,10,11,12Some studies showed the effect of rhEGF eyedrops in vitro and animal studies ,13. HoweFifty subjects (twenty-five each in the single ascending dose (SAD) and the multiple ascending dose (MAD) study) were enrolled. A total of 48 subjects completed this study as planned. Two subjects (one each in SAD and MAD study) dropped out due to the withdrawal of their own consent. There were no significant differences in demographic characteristics among the dose groups in SAD and MAD studies, respectively .p-value 0.7660. Fisher’s exact test) and MAD  studies, respectively. rhEGF eyedrops were well tolerated in all dose groups in SAD and MAD studies. There were six adverse drug reactions (ADRs) by five subjects treated with rhEGF eyedrops and two ADRs by two subjects treated with a placebo in SAD study, and six ADRs by five subjects treated with rhEGF eyedrops and five ADRs by four subjects with a placebo in MAD study, respectively . All ADRThere were no clinically significant findings for vital signs, physical examinations, and ECG. In addition, there were no changes in ophthalmic examinations after the administration of rhEGF eyedrops .max) of tear at ranges of 0.18–0.2 and 0.18–0.27 h in SAD and MAD studies, respectively (max) of tear and the area under the concentration-time curve from the pre-dose to 12 h (AUC0-12h) increased by dose in SAD and MAD studies. However, The observed serum concentration after 12 h of the administration (C12h) of tear was not changed by rhEGF eyedrops in SAD and MAD studies.After the administration of rhEGF eyedrops, the mean serum EGF concentration showed numerous peaks, but it did not change compared to those of baseline and placebo, while the mean tear EGF concentrations increased compared to those of baseline and placebo in SAD and MAD studies . rhEGF eectively . Then, tAfter the administration of rhEGF eyedrops, no anti-EGF antibodies were detected in SAD and MAD studies. Although anti-EGF antibodies were detected in a subject in the placebo group before the administration of rhEGF eyedrops, they were not detected after the administration of the placebo. .This study was the first-in-human study to evaluate the safety/tolerability and PK of rhEGF eyedrops after single and multiple administrations in healthy male subjects. There were some published EGF eyedrops in corneal injury including dry eye syndrome; however, there have been no PK studies of EGF eyedrops . TherefoOne of the primary goals of first-in-human study was to determine safety/tolerability. Considering the safety profiles, rhEGF eyedrops at doses ranging from 10 μg/mL to 100 μg/mL in SAD and MAD studies were well tolerated. No subjects experienced serious adverse events. Among subjects who received rhEGF, corneal erosion was the most common ADR. The incidences of corneal erosion were similar among dose groups, including placebo. In addition, corneal erosions occurred on the medioinferior side, in which tear sampling was commonly conducted. Therefore, corneal erosion may be caused by tear sampling procedures rather than rhEGF eyedrops. After administrations of rhEGF eyedrops, there were numerous peaks in time-serum EGF concentration profiles. Serum EGF levels show high variability through numerous causes, including circadian rhythm, ultradian rhythm, and venous puncture ,16. In aIn this study, tear EGF was rapidly absorbed; then, tear EGF concentrations became similar compared to those of baseline and placebo after 4 h without accumulation. The rapid decline of tear EGF concentrations may result from precorneal fluid drainage, including nasolacrimal drainage and blinking despite the lack of clear mechanisms of rhEGF binding to ocular tissue . FurtherThere has been no published data on therapeutic tear EGF concentrations for corneal injury in humans. Some in vitro studies reported that the therapeutic EGF concentrations were 1–10 μg/L ,23. HereHere, tear EGF exposure did not show dose-linearity although tear EGF exposure increased by dose. The amount of tears may affect the tear EGF concentration, which is thought to have influenced the PK variability of EGF after administration of rhEGF eyedrops. Considering the PK variability and lack of accumulation of rhEGF eyedrops, tear EGF concentration is not thought to be a suitable biomarker for the evaluation of the efficacy of rhEGF eyedrops.Although rhEGF eyedrops have been emerging as a good treatment option for corneal injury, no appropriate dose of rhEGF eyedrops has been known. There are some studies showing that higher doses of EGF eyedrops might be inappropriate for treating corneal injury because of the possibility of auto-inhibition at a high dose of EGF; however, these studies did not show any dose-tear EGF concentration relationships ,24,25. TIn this study, no anti-EGF antibodies were detected except for a subject on a placebo before rhEGF administration. Generally, there are numerous soluble proteins which bind with EGF, including human EGF receptor 1, human EGF receptor 2, and arginine esterase ,27,28. TThere are some limitations in this study. This study was performed on healthy subjects to minimize confounding factors that could influence the study results. Further studies for corneal injury patients are needed to evaluate the efficacy of the drug. In addition, this study was not conducted for female subjects. Generally, the first-in-human studies for healthy subjects were performed for male subjects because the reproductive toxicity of investigational products has not been sufficiently characterized at early development stages. Therefore, this study was conducted for only male subjects, considering the reproductive toxicity of rhEGF eyedrops had not been sufficiently revealed. There were missing values of tear PK sampling due to the difficulty of the sample collection. Nevertheless, the PK profile could be observed in all dose groups.This study was conducted at the Seoul National University Bundang Hospital (SNUBH). The study protocol was approved by the Institutional Review Board of SNUBH (No. B-1902/522-005). This study was conducted according to the major ethical principles of the Declaration of Helsinki and Good Clinical Practice Guidelines. All subjects provided their written informed consent before any study-related procedures.This study was a phase 1, randomized, double-blind, placebo-controlled, SAD and MAD study. The subjects were enrolled and randomly assigned to the rhEGF eyedrops  or placebo in a 3:1 ratio. The subjects received rhEGF or placebo twice a day for one day and 14 days in the SAD and the MAD studies, respectively. The subjects who were assigned to the rhEGF eyedrops received rhEGF eyedrop in one eye, which was randomly assigned, and a placebo in the other eye. Dose escalation was determined based on the evaluation of safety/tolerability data from the previous dose groups. In addition, whether to proceed to the MAD study was determined based on the safety/tolerability results of the SAD study.2 and were 19–50 years of age were eligible for enrollment in the study if no clinically significant abnormalities were observed in medical history, physical and ophthalmic examinations, clinical laboratory tests, vital signs, and 12-lead electrocardiograms (ECG).In this study, healthy male subjects who had a body weight of 50–100 kg and body mass index of more than 18.0 kg/mFor PK sampling, serial blood and tear samples of EGF were collected at the scheduled times: pre-dose and 0.25 h, 0.5 h, 1 h, 2 h, 4 h, and 12 h at −1 d and 1 d, and pre-dose and 12 h at 2 d after dosing for dose groups of SAD study; pre-dose and 0.25 h, 0.5 h, 1 h, 2 h, 4 h, and 12 h at −1 d, 1 d, and 14 d, pre-dose at 2 d, 8 d, and pre-dose and 12 h at 15 d after dosing for dose groups of MAD study. Tear samples were collected from the marginal tear strip of the lower lid near the medial canthus by using disposable microcapillaries. Serum and tear concentrations of EGF were analyzed by a validated enzyme-linked immunoassay (ELISA) using a human EGF Quantikine ELISA kit . Serum and tear samples were diluted appropriately in the Assay Diluent RD1-6. EGF calibration standards were prepared at the following concentrations: 3.91, 7.81, 15.6, 31.3, 62.5, 125, and 250 pg/mL. 200 μL of diluted sample or standard was added to the plate and incubated for 2 h at room temperature. After the incubation, the plates were washed three times with the wash buffer. Next, 200 μL of human EGF conjugate was added to each well, then, incubated for 2 h at room temperature. After the incubation, the plates were washed three times with the wash buffer. After that, 200 μL of the substrate was added to each well and incubated for 20 min at room temperature. Then, 50 μL of reaction termination solution was added to each well to terminate the reaction. The subsequent absorbance was quantified by measurement at 450 nm using a VersaMax Microplate Reader  and the results were analyzed using Softmax Pro 7.1 GxP .® WinNonlin® software version 8.0 . The observed serum concentrations and times were used to estimate the Cmax and C12h of serum and tear EGF and Tmax. AUC0-12h was calculated using the linear up/log down trapezoidal method.Noncompartmental analysis was performed to calculate the PK parameters of serum and tear EGF using PhoenixAnti-EGF antibodies were determined for assessment of immunogenicity from serum samples at the scheduled times; prior to following the rhEGF eyedrops administration at week 4 in the SAD study and prior to following the rhEGF eyedrops administration at weeks 1, 2, and 6 in the MAD study. The anti-EGF antibodies were determined using a validated ELISA. The confirmation of specificity used a floating cut point.Safety/tolerability evaluations including adverse events (AEs), physical examinations, clinical laboratory tests, vital sign measurements, and ECG were conducted In addition, ophthalmic examinations including best-corrected visual acuity, intraocular pressure, refractive error, slit-lamp examination, fundus examination, tear break-up time test, Schirmer’s test, and ocular surface disease index (OSDI) were conducted.In conclusion, the dose range from 10–100 μg/mL solution of rhEGF eyedrops in single and multiple administrations was safe and well-tolerated in healthy male subjects. All ADRs were mild and transient. rhEGF eyedrops increased tear EGF level without effect on systemic exposure. Our findings justify further evaluation of the efficacy and safety of rhEGF eyedrops for corneal injury patients.


Ferroptosis, a novel mode of apoptosis has recently been shown to be associated with fibrosis, tumor, cardiovascular, and other diseases. In this study, using bioinformatic analysis, we identified ferroptosis genes associated with Crohn’s disease (CD) and performed biological function analysis, identified potential drug targets, and provided new directions for the future treatment of CD.Differential expression analysis was performed using the GSE186582 dataset from the Gene Expression Omnibus (GEO) database. Ferroptosis-associated genes were downloaded from the FerrDB database, and overlapping genes associated with CD and ferroptosis were extracted. Then, we performed functional enrichment analysis, constructed a protein-protein interaction network (PPI), identified the correlation between hub genes and immune infiltration, performed external validation using a second and third dataset , and identified potential therapeutic agents. Finally, we validated the protein expression levels of the identified hub genes by immunohistochemical staining in the colon tissues from CD and healthy participants.p < 0.05), and all AUC values were greater than 0.8. Further, we predicted the top 10 drugs used to treat CD. Immune infiltration results suggest that Hub gene is related to T cells, macrophages, dendritic cells, and other immune cells. Finally, the results of immunohistochemical experiments showed that the protein expression of the hub gene was higher in CD colon tissue than in normal subjects (p < 0.05).A total of 28 ferroptosis-associated genes associated with CD were identified in our analysis, which included 22 up-regulated and 6 down-regulated genes. Gene Ontology (GO) analysis showed that these genes are essential for the apical plasma membrane and amide transport, and Metascape analysis showed that these genes mainly act on IL-4 and IL-13 signaling pathways. Five hub genes, PTGS2, IL6, IL1B, NOS2, and IDO1, were identified by a protein interaction network, and external validation of these hub genes showed statistically significant differences in expression between the CD patients and normal participants (Bioinformatics analysis showed that ferroptosis is closely related to the development of CD, and the prediction of potential drugs provides new targets for the treatment of CD. Moreover, five hub genes identified are potentially new and effective markers for the diagnosis of CD. Inflammatory bowel disease (IBD) includes two subtypes, namely ulcerative colitis (UC) and Crohn’s disease (CD). CD is a chronic gastrointestinal disease that can affect all segments of the gastrointestinal tract. At the stage of diagnosis, the majority of the patients have manifestations of intestinal inflammation with typical symptoms such as abdominal pain, chronic diarrhea, and weight loss. As the disease progress, fibrosis can occur leading to narrowing of the intestinal lumen, intestinal fistulas, and abscesses. In severe cases of CD surgical treatment may be required, and extraintestinal complications including anemia, osteoporosis, thrombosis, and even cancer might occur –3. HowevFerroptosis is an iron-dependent, novel form of programmed cell death that is distinct from apoptosis, cellular necrosis, and cellular autophagy. The key factors that trigger ferroptosis are intracellular iron and ROS accumulation, glutathione depletion, and impaired lipid peroxide metabolism , 8. InteCurrently, there is no research on the mechanism of ferroptosis gene in CD based on bioinformatics. In this research, the public dataset was analyzed, the hub genes were screened out and drug prediction was carried out, to explore the mechanism of this new type of apoptosis in CD. The purpose is to find the genes and pathways related to CD and ferroptosis, and to find new therapeutic drugs, thus providing new ideas for the diagnosis and treatment of CD.The study was conducted strictly in accordance with the principles of declaration of Helsinki and was approved by the Ethics Committee of Heilongjiang Hospital .n = 196), and healthy individuals (n = 25).In this study, the dataset GSE186582 was downloaded from the Gene Expression Omnibus (GEO) database to study CD gene expression profile changes. The gene expression profile in this dataset was constructed based on the GPL570 [HG-U133_Plus_2] Affymetrix Human Genome U133 Plus 2.0 Array platform. The dataset contains colon tissue samples from CD patients , the two R packages FactoMineR and factoextra were used and the LIMMA package was used for differential gene expression analysis. Finally, the genes meeting the criteria such as 1 to generate Venn diagrams between differentially expressed genes and the ferroptosis-associated genes. Subsequent analyses included differential genes that overlapped with ferroptosis-associated genes. The heat map of the overlapping differentially expressed genes was also plotted using the R software.Ferroptosis genes were downloaded from the FerrDband Venn diagrams were plotted using the online toolGene Ontology (GO) functional enrichment analysis of differential genes associated with ferroptosis was performed using the GO plot package in R. GO analysis included biological process (BP), cellular component (CC), and molecular function (MF). In addition, the obtained differentially expressed genes associated with ferroptosis were subjected to functional enrichment analysis using Metascape, and these genes were uploaded to the Metascape online analysis tool for annotation of their biological processes.2 and analyzed using STRINE’s online database to predict the interaction relationships between proteins encoded by the genes that may play important role in the pathogenesis of CD. Interaction scores > 0.4 were considered significant and their results were downloaded and saved locally for subsequent visualization. The results were visualized using Cytoscape V3.9.0 software and the key top 5 genes were calculated using the cytoHubba plugin.Differentially expressed genes associated with ferroptosis were uploaded to the STRINE online databaseBased on transcriptome data, single sample gene set enrichment analysis (ssGSEA) algorithm was used to evaluate the immune cell infiltration of patients with Crohn’s disease in GSE186582 dataset. Subsequently, the correlation analysis between the expression levels of 5 core genes and immune cells was carried out. The above analyses were completed by limma package, GSVA package and GSEABase package.For validation studies a second dataset GSE102133 constructed based on the GPL6244 [HuGene-1_0-st] Affymetrix Human Gene 1.0 ST Array [transcript (gene) version] platform was downloaded from the GEO database. This dataset contains samples from 65 CD patients and 12 normal human subjects. The expression differences between the two groups were verified, the working characteristic curves (ROC curves) of hub genes were plotted with R to determine the cut-off values and the area under the curve (AUC) was calculated to assess the clinical diagnostic significance of key genes. In addition, GSE95095 was downloaded from GEO database, which contains 48 patients with Crohn’s disease and 12 healthy people, so as to further verify the differential expression of Hub gene between CD and healthy people.The Connectivity Map (cMap) database is an online database widely used in pharmacogenomic studies to reveal functional associations of small molecule compounds, genes, and disease states. Correlation scores are obtained based on the enrichment of differentially expressed genes in the reference gene set, with values ranging from −100 to 100, with positive numbers indicating a positive correlation with the reference gene set and negative numbers indicating a negative correlation. In our study, small molecule compounds with correlation scores <−80 were used as promising predicted results.Immunohistochemical staining was performed to validate the differential expression of relevant hub genes in the colon tissues of CD patients and normal subjects. Clinical samples were obtained from archived tissues of 10 patients diagnosed with CD who underwent colonoscopy at the Heilongjiang Provincial Hospital. Similarly, 10 archived colon tissues from healthy participants who underwent colonoscopy. Three diseased and three healthy human tissue specimens were selected for each hub gene. Briefly, five sets of 3-mm paraffin sections were prepared and dried (3V3) and dewaxed using xylene solution and ethanol solution. This was followed by heat induced antigen retrieval and blocking using citrate buffer and endogenous peroxidase, and the slides were then rinsed twice using PBS buffer. IL6 , IL1B , NOS2 , PTGS , IDO1  primary antibodies were incubated overnight at 4°C. The sections were then incubated with the secondary antibodies at room temperature for 20 min, washed three times with PBS buffer, and each section was subsequently stained with 3,3′-diaminobenzidine and then re-stained with hematoxylin. Three 200× high magnification fields were randomly observed for each index, and the mean optical density (MOD) values were calculated using Image Pro Plus (IPP) software.t-test, and correlation analysis were calculated using Spearman analysis. P < 0.05 was considered statistically significant .The data were analyzed and visualized using R 4.1.0 and SPSS 26.0 statistical software. Differences in hub genes expression in CD and healthy samples were calculated using the p < 0.05 and |log2 FC| > 1) in the colon of CD patients tissues compared with normal human colon tissues, of which 316 genes were up-regulated and 333 genes were down-regulated as shown in the volcano plot ; amide transport, ; heme binding, and tetrapyrrole binding (molecular function) . FurtherNext, we constructed a network graph using the protein-protein interaction database of the STRING online tool to investigate the role of ferroptosis-associated genes, which contained 28 nodes and 45 edges. In the String database, nodes denote genes, edges denote gene interactions, and the network is set to default as of value (interaction score > 0.4). These 28 genes were analyzed using Cytoscape to construct a network graph, in which 3 genes did not form any network with other genes and were not associated . The topIn order to further understand the correlation between Hub gene and immune cell infiltration, ssGSEA analysis was carried out. The results showed that the expressions of IDO1, IL1B, IL6, and PTGS2 were positively correlated with the infiltration amount of aDCs, B cells, DCs, Macrophages, Neutrophils, NK cells, pDCs, Tfh, Th1 cells, Th2 cells, TIL, and Treg. NOS2 was negatively correlated with mast cells and T helper cells . These rSince we narrowed down the list of key genes associated with ferroptosis and CD, we then looked for potential drugs for these targets. To this end, we used cMap for screening potential pharmacological targets, and the drugs were ranked and identified according to their connectivity scores. The results showed that the top ten drugs used for the treatment of CD were: treprostinil, CG-930, mexiletine, ritanserin, LY-2140023, RS-102221, GW-501516, chenodeoxycholic-acid, tyrphostin-AG -1295, neurodazine .p < 0.0001) in the GSE102133 dataset validating our results    indicatiIn this study, first of all, the differential genes between CD and healthy people is obtained through analysis, subsequently, the differential expression genes related to ferroptosis in CD through screening is obtained, including 22 up-regulated genes and 6 down-regulated genes. PTGS2 is one of the marker genes of ferroptosis, indicating that ferroptosis is involved in the pathogenesis of CD. To figure out the pathogenesis of these genes in CD, functional enrichment analysis has been carried out and it is found that these genes are mainly involved in biological processes including apical plasma membrane and amide transport, and it participates in IL-4 and IL-13 signaling pathways. We also made drug prediction for related genes, and treprotinil, CG-930 and other drugs may become potential drugs for CD treatment. Furthermore, the reliability of bioinformatics analysis is confirmed through the results of external data sets and immunohistochemical validation, providing new ideas for the study of CD pathogenesis and clinical treatment.Although we did not detect differential expression of GPX4 in our bioinformatics analysis, it is speculated that this might result from the fact that some patients received drug treatment, which affected the results, however, a large number of studies showed that the low expression of GPX4 was closely related to Crohn’s disease. GPX4, a selenoprotein, is a major enzyme that catalyzes the reduction of phospholipid hydrogen peroxide in cells. It can reduce peroxide into corresponding alcohol, inhibit the level of peroxidation in cells, and is of crucial importance to ferroptosis , 17. Accvia receptor-mediated endocytosis and catabolized intracellularly . IL-6R can exist as membrane-bound (mIL-6R) or soluble (sIL-6R), of which sIL- 6R is believed to be the main pathway that triggers signaling. IL-6 and IL-6R can activate the gp130 subunit on the cell surface, which in turn activates related signaling pathways, such as the JAK/STAT pathway . In a seIn this study, it was found that the infiltration amount of dendritic cells, macrophages, B cells, NK cells, Th1 cells, Th2 cells, and regulatory T cells in patients with Crohn’s disease was increased. After activation, CD4 + T cells differentiated into T cell subsets with different effects, including Th1, Th2, Th17, regulatory T cells (Treg) and follicular helper T cells (Tfh). Th17-mediated immunoregulatory response played an important role in the pathogenesis of CD, and participated in immune response by secreting IL17, IL26, TNF-α, and other cytokines . Among tWe also identified candidate drug molecules that could target the identified ferroptosis-associated genes. Treprostinil is a stable analog of prostacyclin that promotes direct vasodilation of the systemic arterial vascular bed and inhibits platelet aggregation. Studies have shown that treprostinil activates cAMP, thereby inhibiting tumor growth factor (TGF-β1) secretion and platelet-derived growth factor (PDGF-BB)-induced cell proliferation, and has antifibrotic effects in idiopathic pulmonary fibrosis (IPF) . InteresThere are some limitations to this study. First, the clinical sample size collected in this study was small due to objective reasons such as experimental conditions. Further, the validation study was limited to single-center, small-scale immunohistochemical experiments. Finally, the study of the relevant signaling pathways, hub genes, and potential drug treatment effects in animal models was limited to bioinformatics analysis and lacks further experimental validation.In summary, we identified the ferroptosis genes associated with CD using bioinformatics methods and analyzed their pathways, and mechanisms of action to reveal the potential role of ferroptosis in CD and to identify potential therapeutic agents. Our study provides novel targets for the diagnosis and treatment of CD and may be clinically valuable.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/The studies involving human participants were reviewed and approved by the Heilongjiang Provincial Hospital Ethics Committee. The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.XJ and XS designed the experiments. XJ, DY, RL, and SM performed the analysis. XS and DY supervised the study. XJ, YS, and SM wrote the manuscript. All authors contributed to the article and approved the submitted version.