In recent years, there has been increasing interest in providing an enriched environment to broiler chickens. Indeed, many welfare certification companies encourage or require enrichment to be provided. Most of these companies suggest the use of scatter feeding as enrichment material, though there is little scientific evidence to support the implementation of a scatter feeding program. One of the potential benefits of scatter feeding programs may be an observed increase in foraging behavior, and hence overall activity of the birds. This study aimed to understand the impact of scatter feeding on the foraging behavior of broilers. Six groups of broilers were provided with either dried mealworms, whole wheat, shredded cabbage, alfalfa pellets, wood shavings, or no scatter feeding. To maintain the birds’ interest in the enrichment, feed items were only scattered on the first three days of each week. Foraging and feeding behavior were observed via video for one-hour periods immediately after scattering, 2 h later, and 6 h later. Immediately following the scattering of feed items, broilers in all groups showed an increase in foraging, though this was most pronounced in the dried mealworm group. Foraging behavior decreased with age for all groups. The mealworm group also fed less during hour one compared to the later hours. These results did not provide evidence that scatter feeding encourages foraging behavior, except for a short-term effect of a high value food item. Therefore, future studies should examine the feed item and delivery in more detail.p = 0.001) and active (p = 0.001) foraging, though this was most pronounced in the dried mealworm group. Across all groups, active foraging decreased with age (p = 0.001). The mealworm group also showed a corresponding decrease in feeding during hour one compared to the later hours (p = 0.001). These results did not provide evidence that scatter feeding encourages foraging behavior, except for a short-term effect of a high value feed item. This finding suggests that the item scattered and the delivery method should be studied further.In recent years, welfare certification companies have encouraged the use of scatter feeding as enrichment material, though there is little scientific evidence to support a scatter feeding program. This study aimed to understand the impact of scatter feeding on the foraging behavior of broilers. One hundred eighty Ross 308 chicks were allocated into six treatment groups (six replicates/treatment). Broilers were scatter fed dried mealworms, whole wheat, shredded cabbage, alfalfa pellets, wood shavings, or no scatter feeding, respectively. Enrichment was provided on the first three days of each week. Total foraging, active foraging, and feeding were observed for one-hour periods immediately after scattering, 2 h later, and 6 h later. In all groups, broilers increased both total ( Environmental enrichments are modifications of the environment that aim to promote the performance of normal behaviors and/or animal health. In broiler chicken production, environmental enrichment is often provided with the goal of increasing overall activity, and preventing the development of lameness, hock burns, and breast blisters . In the The idea that the scattering of whole grains or other feed items can be used as a form of environmental enrichment for broilers is grounded in the assumption that foraging behavior is an important part of the normal behavioral repertoire of broilers. For example, a welfare certification organization  states tThe study was conducted over six weeks between June and July 2018 at the Hopkins Avian Facility, University of California, Davis, with approval from the UC Davis Institutional Animal Care and Use Committee .One hundred ninety-five mixed-sex day-old Ross 308 chicks were reared on wood shavings. The chicks were acquired from a commercial hatchery, and individually marked with food coloring on day one of age. Chicks were colored with 5 colors, grouped into 5 pens by color and brooded for five days under ceramic heating bulbs in six identical pens (3.05 m × 1.52 m). A sixth group of 15 extra chicks were not colored. During brooding, chicks had ad libitum access to water and commercially supplied starter feed delivered in a standard 3.5-gal (13.25 L) waterer and 30 lb (13.61 kg) round feeder, respectively. For the first three days, chicks received 23 L:1 D hours light: dark. Daylight hours were subsequently reduced to 20 L:4 D. On day six of age, chicks were placed into 36 pens  chicks were distributed as evenly as possible across the pens. An opaque tarp was hung across pen partitions to prevent chicks in one group from seeing those in adjacent pens. Ad libitum access to water and commercially supplied starter (1 week), grower (2 weeks), and finisher (2 weeks) diets continued to be provided. Researchers and staff entered the barn only to conduct daily wellness and equipment checks, and to clean and refill feeders/waterers.Each of the thirty-six pens was assigned to one of six treatments (six replicates/treatment): (1) dried mealworms (MW), 2) whole wheat (WW), 3) cabbage (CA), (4) alfalfa pellets (AP), (5) wood shavings (SH), and 6) feed-only/no-scattering control (Control). Treatments were assigned in blocks to ensure that they were uniformly distributed across the barn. Enrichments were scattered evenly across the pen floor on the first three days of each week between 10:00 and 11:00 h. Birds in the MW, CA, AP, and SH treatment pens received a half cup (118.3 mL) of enrichment. Due to the difference in grain size (grain units), only ¼ cup (59.15 mL) of WW was used. The Control group served as a non-scatter-feeding control. Specifically, we tested how the scattering of whole wheat, cabbage, alfalfa, dried mealworms, broiler feed, and shavings impacted the feeding and foraging activity of broiler chickens. Due to the fact that the proposed benefits of scatter feeding are linked to increased activity and load placed on the legs, we further differentiated between active and inactive foraging, where active foraging was performed while the bird was standing up or walking. The whole wheat and cabbage treatment were selected for evaluation as they are currently listed as effective forms of enrichment by one or more broiler welfare assurance programs ex.  were similar during H2 and H3 irrespective of treatments . Whereas the percent of observations spent foraging during H1 was increased by only few percent in Control and Other birds , MW birds were foraging during 52.8  % of the observations in H1.The percentage of observations spent foraging was higher immediately after the scattering was provided (H1) than later in the day (H2 and H3) in all treatments, but this pattern was most pronounced in the MW treatment (p = 0.001), though the effect was small .The percent of observations spent foraging was further affected by age (p = 0.001). Whereas birds were foraging actively to a low percent irrespective of treatment during H2 and H3 , the percent of observations spent foraging actively was increased by a few percent during H1 in Control  and Other . MW birds, on the other hand, spent 36.6  % of the observations during H1 foraging actively.Similar to the percentage of observations spent foraging in total, the percentage of observations spent foraging actively was higher immediately after the scattering was provided (H1) than later in the day (H2 and H3) in all treatments. Again, this pattern was most pronounced in the MW treatment (p = 0.001). Birds were foraging actively in 11.5  % of the observations at 2 weeks of age, in 6.6  % of the observations at 4 weeks of age, and in 2.5  %) of the observations at 6 weeks of age.In addition, the percentage of observations spent foraging actively decreased with increasing age (p = 0.001). More specifically, Mealworm birds were feeding in 8  % of the observations during H1, which was lower compared to all other observations .The percentage of observations spent feeding at the feeder was similar across all observations, but birds in the Mealworms treatment were observed feeding less during H1 (p = 0.001), though the effect was small .The percentage of observations spent feeding at the feeder was further affected by age  and only directly after distribution. Despite not being able to modify foraging behavior in the long term, there is a chance that, with the appropriate enrichment, delivery method, and schedule, broiler activity can be increased. It may also be of interest to look at the entire behavioral repertoire, instead of only foraging, as scatter feeding may have effects outside of the measures in the current study.It is recommended that broilers are provided with high-value feed items that will motivate foraging behavior and general activity. Suggested environmental enrichment that promotes locomotor activity, such as mealworms, bales of straw, feed pellets, and various light intensities, may be used in order to increase activity. Enrichment has been found to effectively induce broiler activity when used in combinations. For example, straw bales and light intensity show promising results in promoting activity, which can lead to a reduction in lameness . As enri


To identify key genes involved in vascular invasion in hepatocellular carcinoma (HCC), to describe their regulatory mechanisms, and to explore the immune microenvironment of HCC.n = 373) and GEO data (GSE149614). The key regulatory networks, transcription factors and core genes related to vascular invasion and prognosis were explored based on the CE mechanism. Survival analysis and gene set enrichment were used to explore pathways related to vascular invasion. Combined with single-cell transcriptome data, the distribution of core gene expression in various cells was observed. Cellular communication analysis was used to identify key cells associated with vascular invasion. Pseudo-temporal locus analysis was used to explore the regulation of core genes in key cell phenotypes. The influence of core genes on current immune checkpoint therapy was evaluated and correlations with tumor stem cell scores were explored.In this study, the genome, transcriptome, and immune microenvironment of HCC were assessed by using multi-platform data from The Cancer Genome Atlas (We obtained a network containing 1,249 pairs of CE regulatory relationships, including 579 differential proteins, 28 non-coding RNAs, and 37 miRNAs. Three key transcription factors, ILF2, YBX1, and HMGA1, were identified, all regulated by HCG18 lncRNA. ScRNAseq showed that HCG18 co-localized with macrophages and stem cells. CIBERSORTx assessed 22 types of immune cells in HCC and found that HCG18 was positively correlated with M0 macrophages, while being negatively correlated with M1 and M2 macrophages, monocytes, and dendritic cells. Cluster analysis based on patient prognosis suggested that regulating phenotypic transformation of macrophages could be an effective intervention for treating HCC. At the same time, higher expression of HCG18, HMGA1, ILF2, and YBX1 was associated with a higher stem cell score and less tumor differentiation. Pan cancer analysis indicated that high expression of HCG18 implies high sensitivity to immune checkpoint therapy.HCG18 participates in vascular invasion of HCC by regulating macrophages and tumor stem cells through three key transcription factors, YBX1, ILF2, and HMGA1. Hepatocellular carcinoma (HCC) is a highly aggressive malignant tumor with high mortality . The proVascular invasion, present in 25–50% of HCCs , dividedPrevious studies have assessed clinical risk factors and genetic characteristics of HCC vascular invasion . AlthougIn this study, we comprehensively analyzed the genomic and transcriptome characteristics of HCC as well as the tumor immune microenvironment. A regulatory network based on a CE mechanism has been established. Combined with transcription factor analysis, three key proteins, YBX1, ILF2, and HMGA1, involved in vascular invasion were identified, and gene set enrichment analysis (GSEA) of their regulatory networks showed that all three were regulated by HCG18. When HCG18 expression was high, activation of the vascular endothelial growth factor (VEGF) pathway increased. Single cell sequencing showed that macrophages and tumor stem cells are key to vascular invasion. Macrophage subtype analysis showed that expression of HCG18 and vascular endothelial growth factor A (VEGFA) was positively correlated with infiltration of M0 macrophages and negatively correlated with M1 and M2 macrophages, monocytes and dendritic cells (DCs). Cluster analysis of immune infiltration into HCC tumors showed that promoting transformation of M0 to M2 macrophages could inhibit vascular invasion and improve prognosis. The tumor stem cell score based on the one-class logistic regression (OCLR) algorithm showed that HCG18 and its regulated transcription factors HMGA1, ILF2, and YBX1 positively correlated with the degree of tumor undifferentiation. Pan cancer analysis indicated that high expression of HCG18 implies high sensitivity to immune checkpoint therapy.The results of our study suggest that HCG18 may be a new target for intervening in vascular invasion and improving prognosis of liver cancer.1, and the clinical data of the corresponding samples was obtained from the CBioPortal website2. The single-cell sequencing data GSE149614 was downloaded from the GEO database. The data used to build the CE regulatory network were downloaded from several websites: miRcode, lncBase3, TargetScan4, miRand5, miRDB, and miRTarBase6. Transcription factor analysis used the KnockTF website7. Immune cell gene expression files required by CIBERSORTx software were downloaded from the CIBERSORT website8. The TIDE score was calculated at http://tide.dfci.harvard.edu. Gepia 2.09 was used for expression correlation and survival analysis. Additional quantitative analysis utilized R and related packages, Cytoscape, and GSEA software.Transcriptome data  was downloaded from The Cancer Genome Atlas (TCGA) databaseClinical information for the samples was analyzed by multivariate Cox survival analysis. Both micro- and macro-vascular invasion were included in the vascular infiltration group. For analysis of the effect of vascular infiltration on overall survival time and disease-free progression of patients with hepatocellular carcinoma, we used R and the R package Survminer, Survival.MicroRNAs are short RNAs with lengths of about 22 nucleotides that can regulate the expression of target genes by competing with mRNA for the same microRNA response elements (MREs). In order to construct the CE regulatory network, the regulatory relationships of miRNA and its targets (miRNA-mRNA or miRNA-lncRNA) were firstly predicted. Then the lncRNA-mRNA regulatory relationships were predicted according to the shared miRNA. At the same time, correlations between the regulatory relationships were calculated to screen the results and improve reliability of the prediction.We filtered the CE-pairs according to the following criteria:(A)LncRNA-miRNA interactions were predicted by miRcode and LncBase , mRNAs targeted by miRNAs were predicted by miRDB (score >75), TargetScan (context percentile >50), and miRanda (energy <−15). The expression of two types of interactions should be negatively correlated.(B)LncRNA-mRNA interactions had to satisfy the following conditions:(a)At least one miRNA was shared.(b)P < 0.05).Expression of LncRNA and mRNA were positively correlated (correlation ≥0 and (c)P < 0.05).A hypergeometric test was performed to assess whether a LncRNA and mRNA share miRNAs ((d)Regulation similarity analysis was performed to check the similarity between expression of miRNAs-LncRNA and miRNAs-mRNA (correlation ≥0.5).P < 0.05), LncRNA (P < 0.05), and miRNA (P < 0.05) was obtained. To identify core proteins, survival analysis of proteins in the regulatory network was performed   –3. TransP < 0.05  databases were considered to have significant differences when GSEA P < 0.05 .The GSE149614 dataset was originally designed for exploring the relationship between microenvironment and tumorigenesis, including the primary tumor, portal vein thrombosis, metastatic lymph node and normal control, with a total of 21 samples from 71,915 cells. Ten tumor samples with a total of 31,491 cells were included in this study. The gene filter condition was satisfied in at least 500 cells, the cell filter condition was satisfied in at least 500 genes, and not more than 10% of transcripts were of mitochondrial origin. After data standardization, dimension reduction was conducted by the UMAP algorithm and clustering. SingleR was used to annotate cells based on the tag genes in each cluster. A violin diagram was used to analyze the distribution of expression levels of HCG18, HMGA1, ILF2, and YBX1 in each subtype of cells.10 uses cell gene expression data as input and simulates cell-to-cell communication by combining the interactions of ligand receptors and their cofactors. After the intercellular regulatory relationships were obtained, the key pathways involved in vascular invasion were screened and the key cells communicating with the endothelium were identified. By combining with the expression distribution of HCG18, HMGA1, ILF2, and YBX1, we explored the cell types regulated by these genes in the process of vascular invasion. Pseudo-temporal trajectory analysis was used to further explore the regulatory mechanism of HCG18, HMGA1, ILF2, and YBX1 at the cellular level.To identify key cells involved in vascular invasion, intercellular communication analysis was performed based on single-cell transcriptome data. CellChatBy analyzing the expression distribution and cell communication of HCG18, HMGA1, YBX1, and ILF2, we concluded that these genes are involved in vascular invasion by regulating the function of mononuclear macrophages and tumor stem cells. To further determine the specific types of cells involved in vascular invasion, we used CIBERSORTx to evaluate the infiltration rate of 22 types of immune cells, including M0, M1, and M2 type macrophages, into the tumor. The relationship between HCG18 and VEGFA expression and the amount of infiltration of various types of macrophages suggested that promoting differentiation of macrophages to the M2 type may be a possible approach to intervening in vascular invasion of HCC and improving prognosis. This process was supported by R and the R package Seurat, Monocle CellChat, and CIBERSORT algorithms.Stem cells have the potential to self-renew and differentiate. Cancer progression involves the gradual loss of differentiated phenotypes and the acquisition of progenitor cell-like and stem-cell-like characteristics. Undifferentiated primary tumors are more likely to cause cancer cells to spread to distant organs, leading to disease progression and poor prognosis. Our study shows that tumor stem cells are involved in vascular invasion in HCC. HCG18, HMGA1, ILF2, and YBX1 were expressed in tumor pluripotent stem cells, and cell communication analysis showed that stem cells were key to vascular invasion. In this study, we used a machine learning OCLR algorithm to calculate the tumor stem cell score for each TCGA-HCC sample by establishing a prediction model for pluripotent stem cell samples (ESC and IPSC) from the PCBC dataset. The relationship between HCG18, HMGA1, ILF2, YBX1, and VEGFA expression and tumor stem cell score was verified at the bulk level.11 to obtain the TIDE rating of each sample. Higher TIDE predictive scores were associated not only with poorer immune checkpoint suppression, but also with poorer survival in patients treated with anti-PD1 and anti-CTLA4.To investigate the effect of HCG18 on immunotherapy, we performed a pan cancer analysis. HCG18 – associated tumors were screened by overall and progression-free survival analyses based on TCGA. The correlation between HCG18 and CD274, PDCD1, CTLA4, MSI, and TMB were analyzed in these tumors. The TIDE algorithm uses a comprehensive analysis of hundreds of different tumor profiles to find biomarkers that can predict the efficacy of immune checkpoint inhibitors, determining whether T cells are failing in immune-hot tumors and whether three types of cells are inhibiting T cell infiltration in immune-cold tumors . RNAseq P < 0.05)  . Analysi < 0.05) . By anal < 0.05) . In gene < 0.05) .P < 0.05 was used for screening of mRNA, lncRNA, and miRNA (P < 0.05), hypergeometric distribution (P < 0.05), and regulation similarity coefficient (COR > 0.5 P < 0.05), which left 1,249 pairs of regulation relationships. Finally, survival analysis of the proteins in the network was carried out to select the regulatory network that incorporated mRNA with survival significance. This process retained 393 pairs of regulatory relationships considered to be key to vascular infiltration of HCC. In order to further explore the core genes, this study focused on transcription factors  dimensionality reduction and clustering, 31 clusters of cells were obtained. The classification core genes were extracted, and 7,078 tag genes were obtained. The cell types were annotated by the SingleR package. The heat map showed that the main cell types were liver cancer cells , monocytes , macrophages , T cells , and NK cells (881) .Expression of HCG18, HMGA1, ILF2, and YBX1 in all kinds of cells was analyzed . ExpressForty-six pathways related to cell interactions were identified by cell communication analysis. Among them, vascular invasion is related to the pro-angiogenesis VEGF pathway and the anti-angiogenesis VEGI  pathway. Mononuclear macrophages and tumor stem cell-like cells in the VEGF pathway are the core cells , while cWe evaluated the infiltration score of 22 immune cells in TCGA samples and analyzed their correlation with expression of HCG18 and the transcription factors HMGA1, ILF2, and YBX1 . The resP = 0.12), which may be related to the low basal expression level of HCG18.The above analysis showed that HCG18, HMGA1, ILF2, and YBX1 could participate in vascular invasion via tumor stem cells. We evaluated stem cell scores in TCGA patients using the OCLR algorithm  and perfP value < 0.05)  . HCG18 w < 0.05) . As showHepatocellular carcinoma (HCC) is one of the most common complex diseases in the context of chronic liver disease. Over the past 20 years, our understanding of the clinical and molecular heterogeneity of HCC has improved dramatically. The most commonly used clinical classification scheme is the Barcelona Clinical Liver Cancer (BCLC) system , which dIn this study, we mined three key transcription factors, YBX1, ILF2, and HMGA1, by constructing a vascular invasion-related CE regulatory network that included transcription factors.YBX1 encodes a highly conserved cold-shock domain protein that binds to a wide range of nucleic acids, including both DNA and RNA, and mainly acts as an RNA binding protein to recognize mRNA transcripts modified by C5-methylcytosine (M5C) . mRNA stThree transcription factors, YBX1, ILF2, and HMGA1, have all been shown to be involved in vascular dysplasia, and our study also shows that they play a key role in vascular invasion. The CE regulatory network revealed that YBX1, ILF2, and HMGA1 were all regulated by HCG18. Therefore, HCG18 is believed to play a key regulatory role in vascular invasion. RNASeq and scRNASeq showed that overall expression of HCG18 was not high in tumors, but was specifically distributed in mononuclear macrophages and tumor stem cells, which were identified as being key to vascular invasion based on cellular communication analysis. Correlation analysis of HCG18 expression at the bulk level and immune infiltration showed that HCG18 expression was positively correlated with M0 macrophage infiltration, although negatively correlated with infiltration of M1 and M2 macrophages and monocytes. Cluster analysis based on immune infiltration showed that the proportions of M0, M1, and M2 macrophages affected prognosis. When the proportion of M0-type macrophages increased and the proportion of M2 macrophages decreased, the expression of VEGFA increased and prognosis was poor. The pseudo-time trajectory suggested that HCG18 and its regulated transcription factors can regulate the phenotypic transformation of macrophages.In recent years it was found that the immune microenvironment in tumors plays an important role in regulation of angiogenesis. Angiogenesis is mainly related to the secretion of VEGF, TGFβ and other cytokines by immune cells that infiltrate tumors, and tumor-associated macrophages play a key role in this process . Based oTumor-associated macrophages (TAMS) are usually the major component of myeloid cells in tumors. Macrophages have been shown to be directly or indirectly involved in several key features of malignancy, including angiogenesis, invasiveness, metastasis, regulation of the tumor microenvironment, and therapeutic resistance . This maImmunotherapy is considered to be the most promising cancer treatment. A variety of cancer immunotherapies, including adoptive cell immunotherapy, tumor vaccines, antibodies, immune checkpoint inhibitors, and small molecule inhibitors, have had some success . TherapeOur study shows that high expression of HCG18 implies high sensitivity to immune checkpoint therapy. Compared with the currently known CD274, PDCD1, CTLA4, MSI, and TMB, HCG18 is not a direct target of immunotherapy, and as a non-coding RNA, its basic expression level is low. However, HCG18, as a non-coding RNA, increases the evaluation dimension of immunotherapy. Meanwhile, our study showed that HCG18 can be used as an evaluation indicator for vascular invasion, while other currently known immune evaluation indicators have no clear relationship with vascular invasion. In conclusion, HCG18 can simultaneously evaluate tumor angiogenesis, invasion and immune microenvironment.The results of our study suggest that HCG18 should be a new target for treatment of hepatocellular carcinoma.The conclusions obtained in this study are based on bioinformatics analysis. The protein expression level of the gene, its corresponding regulatory relationship and its influence on cell phenotypic transformation need to be further explored experimentally.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/LZ, HB, JG, and ZJW: conceptualization. LZ: methodology, software, formal analysis, and original draft preparation. ZWW, ML, and PS: validation. TB: investigation and resources. WW: data curation. JG and ZWW: review and editing. LZ and PS: visualization. ML: supervision. HB: project administration and funding acquisition. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.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.


Background: Late-onset Alzheimer’s disease (LOAD) and early-onset Alzheimer’s disease (EOAD) are different subtypes of AD. This study aimed to build and validate radiomics models of the hippocampus for EOAD and young controls (YCs), LOAD and old controls (OCs), as well as EOAD and LOAD.Methods: Thirty-six EOAD patients, 36 LOAD patients, 36 YCs, and 36 OCs from the Alzheimer’s Disease Neuroimaging Initiative (ADNI) database were enrolled and allocated to training and test sets of the EOAD-YC groups, LOAD-OC groups, and EOAD-LOAD groups. Independent external validation sets including 15 EOAD patients, 15 LOAD patients, 15 YCs, and 15 OCs from Shanghai Mental Health Center were constructed, respectively. Bilateral hippocampal segmentation and feature extraction were performed for each subject, and the least absolute shrinkage and selection operator (LASSO) method was used to select radiomic features. Support vector machine (SVM) models were constructed based on the identified features to distinguish EOAD from YC subjects, LOAD from OC subjects, and EOAD from LOAD subjects. The areas under the receiver operating characteristic curves (AUCs) were used to evaluate the performance of the models.Results: Three, three, and four features were selected for EOAD and YC subjects, LOAD and OC subjects, and EOAD and LOAD subjects, respectively. The AUC and accuracy of the SVM model were 0.90 and 0.77 in the test set and 0.91 and 0.87 in the validation set for EOAD and YC subjects, respectively; for LOAD and OC subjects, the AUC and accuracy were 0.94 and 0.86 in the test set and 0.92 and 0.78 in the validation set, respectively. For the SVM model of EOAD and LOAD subjects, the AUC was 0.87 and the accuracy was 0.79 in the test set; additionally, the AUC was 0.86 and the accuracy was 0.77 in the validation set.Conclusion: The findings of this study provide insights into the potential of hippocampal radiomic features as biomarkers to diagnose EOAD and LOAD. This study is the first to show that SVM classification analysis based on hippocampal radiomic features is a valuable method for clinical applications in EOAD. Alzheimer’s disease (AD), characterized by progressive cognitive dysfunction, is a common neurodegenerative disorder that significantly affects the quality of life of patients  databasen = 36; YC) and an OC group for LOAD . Furthermore, demographic information, medical history, baseline symptoms, and assessment scale scores were included. The MRI and clinical data were downloaded in June 2021.A total of 144 ADNI participants were included in this study 36 EOAD, 36 LOAD, 36 young control (YC), and 36 old control (OC) participants from the ADNI1, ADNI2/GO, and ADNI3 databases. Scans were collected at either screening or baseline visits. First, 36 patients diagnosed with AD onset before the age of 65 years (EOAD) who were enrolled in the ADNI database were eligible for this study. Next, we included 36 patients who were 65 years or older at disease onset (LOAD) and who were 1:1 matched to the EOAD patients by the Clinical Dementia Rating (CDR) Scale. Accordingly, we selected two control groups for each patient group. The controls were matched 1:1 to AD patients for age and sex, thus obtaining a YC group for EOAD  between July 2017 and May 2021, and normal control subjects were recruited from the community. A total of 60 participants including 15 EOAD, 15 LOAD, 15 YCs, and 15 OCs were enrolled in this study. Similarly, 15 LOAD patients were also 1:1 matched to the EOAD patients using the CDR Scale, and the controls were matched to AD patients for age and sex. EOAD and LOAD patients were diagnosed by two experienced geriatric psychiatrists. The exclusion criteria included the following: (1) other psychiatric disorders comorbidities; (2) a history of major physical illness, cardiovascular disease, or neurological disorder; (3) substance abuse or dependence; (4) pregnancy. Neuropsychological tests and brain imaging scans were performed in all subjects. The retrospective study was approved by the ethics committee of the Shanghai Mental Health Centre of Shanghai Jiao Tong University School of Medicine, and all the participants provided written informed consent after they were given a description of this study.time (TR) = 2,300 ms, matrix = 240 × 256 × 176, slice thickness = 1.2 mm, and those parameters in General Electric scanner were as follows: TR = 7 ms, matrix = 256 × 256 × 166, slice thickness = 1.2 mm and those parameters in Philips scanner were as follows: TR = 6.8 ms, matrix = 256 × 256 × 170, slice thickness = 1.2 mm, respectively. More detailed information about the image acquisition procedures is available on the ADNI website3).Regarding ADNI data, T1-weighted structural imaging was collected using a 3D MPRAGE (magnetization prepared rapid gradient-echo imaging) sequence with slightly different MR parameters among participants. The MR images acquired using Siemens scanner were scanned with the parameters as follows: repetition Standardized preprocessing was necessary to improve discrimination between textural features and was performed using Statistical Parametric Mapping (SPM12) softwareSegmentation of the hippocampus was required to describe the texture characteristics of the region of interest (ROI). First, the bilateral hippocampus from the Anatomical Automatic Labeling (AAL) template provided by the MNI was chosen as the ROI mask. Then, the open-source software 3D-slicerFirst, we loaded the standardized 3D T1-MPRAGE data for the EOAD, LOAD, YC, and OC subjects into 3D-slicer software, and then we imported the segmented left and right hippocampus. Massive features were selected using the “pyradiomics” package of the softwareHISTO is a statistical description of discrete units, while the GLCM using second-order statistics reflects the spatial relationship of pixel gray-level values in the image . Next, correlation analysis was performed to further reduce the dimensionality. If the correlation coefficient of two feature columns exceeded 0.8, we removed one of them randomly. Finally, the least absolute shrinkage and selection operator (LASSO) regression analysis method with 10-fold cross validation was applied to determine the most valid features in the training data, and the corresponding lambda value was selected with minimum mean-squared error (MSE) values. The mechanism of LASSO, combining the penalty function and linear regression, makes some regression coefficients become zero and achieve dimension reduction  algorithms were used to construct radiomic models for EOAD and YCs, LOAD and OCs, and EOAD and LOAD. SVM is one of the most popular and mature machine learning algorithms based on the neuroimaging literature  to evaluate the differences between groups. A p < 0.05 was considered statistically significant.Statistical analyses were performed using SPSS software 22.0 . The demographic information of the participants was collected as numbers or means ± SD for categorical and continuous variables. The comparisons between the EOAD and OC (EOAD-OC), LOAD and YC (LOAD-YC), and EOAD and LOAD (EOAD-LOAD) subjects were performed using χp < 0.001). No significant differences were found in the clinical dementia rating (CDR) scores and MMSE scores between the EOAD and LOAD patients (EOAD-LOAD).The demographic and clinical characteristics of the four groups are presented in A total of 214 features were extracted from the bilateral hippocampus. After t-test and Mann-Whitney U test, 99, 102, and 37 features were preserved in the EOAD-YC, LOAD-OC, and EOAD-LOAD groups, respectively. After correlation analysis, 51, 73, and 24  featuresThe accuracy (ACC), sensitivity (SEN), specificity (SPE), and AUC were used to evaluate the classification performance. The present study aimed to explore hippocampal radiomic features to distinguish between patients with EOAD and LOAD and healthy controls. Our findings show that the hippocampal radiomic-based classification model can discriminate patients with EOAD from YC subjects and distinguish LOAD patients from OC participants. Additionally, hippocampal texture was identified as a useful biomarker for LOAD and EOAD patients. Additionally, results from other datasets verified the generalizability and robustness of the models.To our knowledge, this study is the first to construct a classification model of hippocampal radiomic features for EOAD patients and healthy subjects. This model reveals relatively good accuracy and sensitivity with a successful diagnostic value. Although EOAD patients account for 5–10% of reported AD cases , fMRI, diffusion tensor imaging (DTI), and multimodal MRI, to detect structural and functional changes in AD . Kurtosis measures the degree of histogram sharpness, coarseness reflects the spatial rate of changes in gray-level intensities, and IMC1 captures the spatial relationships of pairs of pixels  can be found below: The studies involving human participants were reviewed and approved by Shanghai Mental Health Center. The patients/participants provided their written informed consent to participate in this study. The use of the ADNI data was approved by the institutional review board at each site. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.XL, YT, and YD conceived the study. YD, SZ, YF, QQ, LZ, and WW acquired and analyzed the data. YD and SZ drafted the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.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.


Candida albicans and Streptococcus aureus were treated with spray formulations containing green silver nanoparticles (GS), chemical silver nanoparticles (CS), or pomegranate peel extract (PS). After wound development and infection, the treatments were performed twice per day for 14 days. The wound healing was analyzed on days 2, 7, and 14 through the determination of CFUs, inflammatory infiltrate, angiogenesis, fibroplasia, myeloperoxidase, and collagen determination. Expressive improvement in wound healing was noted using both silver nanoparticles for 7 days. All the treatments were superior to controls and promoted significant S. aureus reduction after 14 days. CS presented better anti-inflammatory results, and GS and CS the highest number of fibroblasts. Despite the techniques’ limitations, GS and CS demonstrated considerable potential for managing infected wounds, especially considering no early strategies prior to the drugs, such as the debridement of these wounds, were included.Infected cutaneous ulcers from diabetic rats with  The skin is the largest human organ that plays an important role in human homeostasis, acting as a physical barrier against pathogen invasions . EventuaBiofilms are very organized and dynamic communities of different microorganism species embedded in an extracellular matrix . In addiRecently, many efforts have been made to develop new agents targeting the healing process that avoids amputation and other complications related to chronic wounds. The search for efficient drugs against microorganisms in both the planktonic state and biofilm structures, besides presenting anti-inflammatory effects, is an open field that can be widely explored . In thisChrozophora tinctoria, Hibiscus rosa-sinensis L., Pongamia pinnata, Persea americana, Chamaemelum nobile L., and Punica granatum [The use of natural extracts applied as antimicrobial drugs is a growing field in science that has demonstrated interesting and promising results ,23. Follgranatum ,28,29,30Additionally, the use of silver nanoparticles (AgNPs) is widely studied and applied, such as with incorporation in shampoos, soaps, detergents, cosmetics, medical devices, and other applications. As with other nanometric materials, AgNPs are ordinarily defined as presenting at least one special dimension in the size range of 1–100 nm . AlthougThe use of silver nanoparticles as antimicrobial agents is increasing in different areas such as in the cosmetic, medical device, and textile industries ,36,37,38Despite the wide application, conventional silver nanoparticles produced through the chemical route, in addition to its high costs, also show relevant side effects, such as high levels of toxicity, to mammalian cells . These sS. aureus and C. albicans. Additionally, this green formulation presented reduced levels of cytotoxicity when compared to the spray formulation containing silver nanoparticles produced through the chemical route [Recently, our group synthesized and evaluated the cytotoxic effects and antimicrobial activity of novel spray formulations based on silver nanoparticles, comparing nanoparticles synthesized through the green or conventional routes . Our resal route . In the S. aureus and C. albicans when compared to AgNPs synthesized through a conventional chemical route [In a previous study, we demonstrated that spray formulations composed by AgNPs synthesized through a green route using pomegranate peel extract, under in vitro conditions, showed higher antimicrobial activity against al route . In addial route . Aiming To obtain more insights about the antimicrobial activity exerted by the target analyzed formulations here, the number of CFUs was directly assayed from animal ulcers after 2, 7, and 14 days after each specific treatment. Although the CS, GS, and PS formulations had shown considerable wound healing activity, no CFU reduction was observed for C. albicans either after treatment with those three formulations or for the C and Sulf groups A. After To further characterize the inflammatory infiltrate promoted by the target formulations , we quantified the inflammatory infiltrate using the histological approach (HE) followed by image quantification after 2, 7, and 14 days of treatment . The SQ The triggering of fibroplasia is a hallmark of wound healing progression. Due to this, we evaluated the fibroplasia levels induced by the application of spray formulations CS, GS, PS, and Sulf C. After Wound healing in diabetic individuals could be improved by the treatments that promote an increase in blood vessels. Here, we investigated if the pulverization of CS, GS, and PS formulations, and Sulf would be important to increase the number of blood vessels . AccordiMPO is an enzyme that belongs to the heme peroxidase superfamily. This enzyme is produced by neutrophils and is biochemically used to measure the inflammatory process. Aiming to evaluate the inflammatory process triggered by GS, CS, PS, and Sulf, we measured the production of MPO from ulcer lesions recovered from the animal groups treated with these formulations. After 2 days of treatment, none of the formulations presented differences in terms of MPO production when compared to the control group . After 7Collagen production is an important event that happens during the wound healing process and skin formation. Due to this, we used an indirect method to assess the collagen deposition through a hydroxyproline measurement, a derivative of proline in the collagen production pathway. After 2 days, treatments with CS and GS sprays increased the levels of hydroxyproline , corroboThe emergence of multidrug-resistant microorganisms is a global concern that is related to the indiscriminate use of commercially available antimicrobial drugs. In addition, the increasing number of patients with chronic diseases, such as arterial hypertension and diabetes, drives the search for new biomaterials that may be used as medicinal drugs. Therefore, considering the increase in the global diabetic population added to the rise in antimicrobial resistance, the current study focused on the characterization of spray formulations containing silver nanoparticles, produced by both chemical and green routes, and a pomegranate peel extract spray. The spray formulations were evaluated in terms of their ability to improve wound healing in diabetic rats with manually infected cutaneous ulcers. Additionally, their effects on fibroplasia, collagen deposition, angiogenesis, inflammatory effects, and antimicrobial activity were evaluated from ex vivo samples. All the treatments were compared with the control (without treatment) and silver sulphadiazine (as a reference standard treatment).The healing process is linked to several biological events that culminate with total wound closure and healing. During this process, re-epithelialization, fibroplasia, angiogenesis, and extracellular matrix production are important steps that are generally dependent on the immunological condition of the host ,46,47,48P. granatum peel extract that demonstrated efficacy in the healing of chronic wounds in rats [Prosopis juliflora leaf extract and silver nanoparticles (PJL–AgNPs), along with Carbopol, promoted wound contraction and accelerated the healing process in mice in comparison to the negative and positive control groups [Here, we demonstrated that the pulverization of chemical or green silver nanoparticles improved the wound healing process in diabetic rats mainly after 7 days treatment. The formulations containing silver nanoparticles (SC and SG) presented the best results when compared to the pomegranate spray formulation without nanotechnology (SP). This can be explained by the fact that AgNPs are efficient in promoting the differentiation and maturation of keratinocytes through the stimulation of skin stem cells . AgNPs c in rats , corrobol groups .The application of the PS formulation without nanotechnology was able to promote the healing process mainly after 14 days of treatment. Corroborating this, a study demonstrated that the use of an ointment containing 15% pomegranate performed about 93% of wound contraction in rats after 16 days of treatment . In addiC. albicans or S. aureus. At a later time point (14 days of treatment), the CS, GS, and PS formulations, and sulfadiazine reduced the number of CFU of wounds infected with S. aureus, suggesting that all these products played antibiotic functions in the evaluated model of skin ulcers. Adibhesami and colleagues (2017) [S. aureus activity of silver nanoparticles applied to the healing process in mice. On the other hand, CS, GS, and PS were not able to statistically reduce the number of CFU of C. albicans; a very similar profile was observed in the group of animals treated with sulfadiazine, suggesting that none of these formulations had anti-Candida activity. Although the results of the antibiofilm action were not so expressive for C. albicans, a study by Paosen and colleagues (2019) [S. aureus and C. albicans and altered the morphological microbial cells, especially shrinking deformation in regard to C. albicans. Additionally, factors such as interkingdom interactions must be taken into account since, as reported by Kean et al. (2017), the biofilm formation of C. albicans and S. aureus favored strengthening both, increasing resistance to fluconazole [C. albicans such as farnesol, a molecule that regulates several functions in the biofilm, including growth control [The antimicrobial and anti-inflammatory properties of pomegranate are due to the presence of polyphenols , in addition to other benefits such as its antioxidant and antiallergic effects ,58,59,60s (2017)  obtaineds (2019)  reportedconazole . The res control .Current strategies for wound management include first cleansing followed by wound debridement . Even wiP. granatum AgNPs  had low cytotoxicity against a human normal cell line (HFB4) after encapsulation into hydrogels, once more elucidating that the use of AgNPs can offer benefits without being cytotoxic [The induction of fibroplasia is an important effect triggered by many compounds that induce the healing process, and it is linked to tissue reparation. Our results suggest that AgNP GS and CS are not cytotoxic and induce a substantial increase in the number of fibroblasts, specifically after 2 days of treatment, further improving the results for CS nanoparticles. Furthermore, at this time point, the indirect method used here to assess collagen deposition, hydroxyproline measurement, demonstrated that these two groups of animals also presented increased levels of this amino acid. This result suggests that, in addition to fibroplasia, collagen deposition was increased in these groups at initial time points. Ragab and colleagues 2019) demonstrated under in vitro conditions that 9 demonstAslam and colleagues (2006)  reportedWhen an injury happens, the inflammation process helps in terms of the removal of damaged cells and the promotion of vasoconstriction . AdditioWe quantified the inflammatory infiltrate of the animal groups treated with our formulations, and after 7 days of treatment, the recruitment of proinflammatory cells was reduced in all the groups, including SG and SC, with the more expressive reduction seen in the SC group. This finding corroborates the previous result obtained for the induction of fibroplasia, implying that in these groups, the pomegranate formulations reduced the inflammatory response process after 7 days treatment. Lastly, after 14 days of treatment, all the groups presented very low levels of inflammatory infiltrate. A recent study explored the anti-inflammatory response triggered by garlic silver nanoparticles (G–AgNPs) through an in vitro model measuring the denaturation of protein (BSA) as an indicator of the inflammatory process . The autThe use of products based on pomegranate extracts has been evaluated in different animal models and also in cell cultures. The application of an ointment containing 10% pomegranate peel extract reduced the inflammatory process in the burn wounds of rats after 15 days . The addPunica granatum peel extract, and 337.5 µg/mL green silver nanoparticles or 5.55 µg/mL chemical nanoparticles.The spray formulations obtained by Fernandes and colleagues (2018) were used in the current work. Briefly, after obtaining the peel pomegranate extract and the synthesis of silver nanoparticles by both the Turkvich methodology ,75 and gAll protocols adopted in this study encompassing animals were approved by the local Ethics Committee for Animals from the Faculty of Medicine of Ribeirão Preto, University of São Paulo, Brazil, (process nº 140/2016)Male Wistar rats (180–200 g) were obtained from the Central Bioterium of the Medical School of Ribeirão Preto, University of São Paulo, Brazil. The animals were kept in isolation conditions in individual cages, with water and feed ad libitum, and alternating cycles of luminosity every 12 h. Animals received streptozotocin (50 mg/kg) intravenously via the caudal artery. After 24 h, blood glucose concentration was checked for diabetic induction .Staphylococcus aureus and Candida albicans, then directly pipetted (100 μL) onto the lesion. The ulcerated skin was collected to be used as control (Day 0) nonulcerated skin. After surgery, oral dipyrone, 50 mg/kg body weight, diluted in saline solution every 12 h for the first 48 h was orally administered according to the behavioral changes of the animals regarding pain.After confirmation of the induction of diabetes, the rats were weighed and anesthetized intraperitoneally with 70 mg/kg ketamine and 12mg/kg xylazine. Later, the rat dorsum was trichotomized and two surgical excisions were performed with a punch of 1.5-centimeter diameter, thus leaving the dermoepidermal region affected. After this procedure, the microorganisms were adjusted to 104 CFU/mL for The animals were divided into 5 groups containing 6 animals each, and analysis was performed at 2, 7, and 14 days after treatments. Each animal was topically treated twice a day, at the beginning of the morning and at the end of the afternoon. The groups were (C) animals that received no treatment; (GS) green spray formulation containing green silver nanoparticles, (CS) chemical spray formulation with chemical silver nanoparticles, (PS) pomegranate spray, and (Sulf) commercial silver sulfadiazine.At the end of the assay, animals were euthanized in a CO2 chamber after 2, 7, and 14 days. The lesions were photographed with a Sony DSC-P100 digital camera coupled to a base containing a millimeter ruler, with a standard distance of 30 cm, perpendicular to the ulcers, and the lesion areas were measured with Image J software. Then, the ulcer healing index was determined, which is equivalent to the quotient of the difference between initial and final areas divided by the initial area .After photo acquisition, the animals had all skin trimmed around the ulcers and through the 1.5-centimeter diameter punch. The recovered materials were collected and packaged in separated tubes. Separated samples were used for the following purposes:Sample 1: tissue was packed into an Erlenmeyer flask containing 4% formaldehyde in Sorensen phosphate buffer 0.1 M, pH 7.3 for histological study .Sample 2: one wound sample of each animal was stored at −70 °C for biochemical analysis (hydroxyproline and MPO assays), and the other wound sample was used to perform microbiological assay.C. albicans, and Mannitol salt agar for S. aureus, both media from Becton, Dickinson, and Company, Heidelberg, Germany. The plates were incubated at 37 °C for 24 h for S. aureus, and 48 h for C. albicans. Colonies were counted, and the values were expressed by tissue weight (CFU/mg).Tissue from Sample 2 (aforementioned) was thawed and packed into 50-milliliter falcon tubes (BD). Further, 3 mL of PBS was added and vortexed under constant stirring for 1 min. After this step, the contents were submitted to serial dilution and plated in a culture medium specific for each microorganism, Candida chromagar for ® DM4000B optical microscope with a LEICA® DFC 280 camera with LAS® software, Leica Application Suite for image capture. Then, 4–5 fields per blade were obtained at 200× and 400× magnification. The CellCounter’ plugin of ImageJ 1.48 software was used to count the cells of the inflammatory infiltrate, fibroblasts, and blood vessels in each of the 5 individually quantified images of each animal. Each cell or structure was differentiated by the observer in the image of the lamina increasing by 400×, whereas the blood vessels were quantified by a 200× increase.After euthanasia, tissue samples from the animals of each treatment were stained using the hematoxylin–eosin (HE) staining method. Histological slides were visualized on a LEICAThis protocol was applied to the animals of each treatment and each time; for the final result, the mean number of inflammatory infiltrates, fibroblasts, and vessels found in the 4–5 fields of each animal was considered. The graphs were plotted with the mean obtained in each group using GraphPad Prism 5 software , along with the presentation of the standard deviation.g, and resuspended in 0.05 M NaPO4 (Sigma-Aldrich) buffer (pH 5.4) containing 0.5% hexadecyltrimethylammonium bromide (HTAB) (Sigma-Aldrich). A total of 25 μL of the supernatant and 25 μL of TMB  (Sigma-Aldrich) were added to each well of a 96-well plate. Subsequently, 100 μL of 0.5 mM H2O2 was added to each well, and the reaction was quenched with 4 M sulfuric acid. MPO activity was measured in a plate reader  at 450 nm. Results are reported as the total number of neutrophils/mg tissue by comparing the absorbance of the tissue supernatant to a standard curve.The neutrophil dosage in the lesion was performed using the myeloperoxidase assay . Tissue ® PT 3100 at 13,000 rpm with 6N hydrochloric acid (HCl) and transferred to capped glass test tubes. HCL ¨N was added in a proportion of 100 μL per 1 mg of dry tissue, homogenized and subjected to acid hydrolysis, thus incubating the samples in HCL (Sigma-Aldrich) at 130 °C for 4 h.Collagen was assessed by the measurement of hydroxyproline in tissue, following the methodology described by Reddy and Enwemeka (1996) , and DwiAfter incubation, the pH of the samples was adjusted to 7.0 with 2M sodium hydroxide (NaOH) (Sigma-Aldrich). Subsequently, 10 μL of each sample along with 90 μL of 0.056 M T-Chloramine solution were pipetted into 96-well plates incubated in the dark for 25 min. Afterwards, 100 μL of Ehrlich reagent  was addeGraphPad Prism software  was employed for statistical analysis with a confidence level of 95%. Parametric statistical analyses were conducted with a one-way ANOVA followed by Bonferroni test.The presented results showed that silver nanoparticle formulations (CS and GS), regardless of the route of synthesis being chemical or green, have considerable potential for the treatment of wounds in a diabetic model, especially promoting significant improvement in terms of healing and wound closure after 7 days of treatment when compared to commercial silver sulfadiazine. For both silver nanoparticle types, the mechanism of action was focused on re-epithelization stimulation. For PS formulation, wound closure was mediated by a proinflammatory stimulus, with similar behavior to that of other natural compounds evaluated in the same model. Considering the reproducibility, low cost, and ecofriendliness of green synthesis, besides other properties that it could present, and the same benefits for the pomegranate spray, the produced formulations must be explored more deeply as potential antimicrobial and wound healing agents.


Background: Few studies to date have addressed global cardiovascular (CV) risk profile in a “protected” young population as that of medical school students. Objective: to assess CV traditional risk factors and global CV risk profile of Italian medical students throughout the six years of university. Methods: A cross-sectional survey accessible online via quick response (QR) code was conducted among 2700 medical students at the University of Milan, Italy. Data on baseline characteristics, traditional CV risk factors, diet, lifestyle habits, and perceived lifestyle variations were evaluated across different years of school. Results: Overall, 1183 students ) out of 2700 completed the questionnaire (43.8% rate response). More than 16% of the students had at least 3 out of 12 CV risk factors and only 4.6% had ideal cardiovascular health as defined by the American Heart Association. Overweight, underweight, physical inactivity, sub-optimal diet, smoke history, and elevated stress were commonly reported. Awareness of own blood pressure and lipid profile increased over the academic years as well as the number of high-blood-pressure subjects, alcohol abusers, and students constantly stressed for university reasons. Moreover, a reduction in physical-activity levels over the years was reported by half of the students. Conclusion and Relevance: This study demonstrates that a “protected” population as that of young medical students can show an unsatisfactory cardiovascular risk profile and suggests that medical school itself, being demanding and stressful, may have a role in worsening of the lifestyle. Hence, dulthood , but alsdulthood .Thus, the Committee on Preventing the Global Epidemic of Cardiovascular Disease recommended that interventions at “all stages of life course” should be performed in order to promote cardiovascular health by preventing acquisition and augmentation of risk. Early health education and early correction of an unfavorable lifestyle can prevent CV diseases in later life . Indeed,Data regarding cardiovascular risk profile among medical students are limited. Medical students may be a “protected” population of young people given the greater awareness they develop during medical school about what the CV risk factors are and how to address them. Thus, they have the tools to fall within a lower CV risk profile. Indeed, there is a well-established inverse relation between education and mortality from cardiovascular disease. This inverse gradient is mediated by a lower prevalence of modifiable CV lifestyle factors  among educated subjects .On the other hand, it is acknowledged that medical school is a long  and demanding path that often limits students’ time and involves high stress, thus pushing students to have unhealthy lifestyle habits ,13. AccoWe performed a cross-sectional survey study through a peer-to-peer administration of an anonymous questionnaire  general data; (2) anthropometric, clinical, and lifestyle data; (3) diet; (4) pharmacological history; (5) perceived lifestyle variations during the six years of medical school (this section was not filled in by 1st year students). The basic demographic characteristics included age, gender, height, and weight. The body mass index (BMI) was automatically calculated in Microsoft Excel using the following formula: BMI = (Weight in kilograms) divided by (Height in meters squared). The BMI values of the students were used to categorize the subjects as underweight (BMI below 18.5), normal (BMI 18.5–24.9), overweight (25.0–29.9), or obese (30.0 plus). In addition to information regarding reported history of high blood pressure (BP), hypercholesterolemia, diabetes mellitus, and CV disease family history, data on smoking habits, alcohol and illicit drug use, sleep time, and physical activity were acquired and defined according to current guidelines. Specifically, European Society of Cardiology (ESC) guidelines were used to evaluate physical activity  general . The num2 < BMI > 24.9 kg/m2, optimal physical activity, and optimal diet) and 3 ideal health factors .The 14-item PREDIMED SCORE, an index of Mediterranean diet adherence, was used to assess nutrition . To limiThe database was formatted through Microsoft Excel software and subsequently imported into IBM-SPSS software ver. 25.0.2. The use of Stata software ver. 15.0 was also used for any comparisons or implementations of test output. For the statistical analyses, the continuously expressed variables were subjected to the Kolmogorov-Smirnov test to evaluate their normality, log-normality, or non-parametric distribution. The statistical investigation path began with the descriptive analysis of the variables and the related calculation of the main-position indices, or composition-ratio percentages. The inferential analyses, both parametric and non-parametric, were conducted using appropriate tests: Spearman’s r, χ squared and the exact Fisher test, Mann Whitney U-test, Kruskal-Wallis H-test. All results were tested at the α-significance level of 5%. The authors take full responsibility for the integrity of the data.Of the total 2700 students enrolled in the medical school at the University of Milan, 1183 (43.8% response rate) eventually completed the questionnaire. The distribution of responses over time shows that 3/4 were generally concentrated during the first week (23–27 September) while the rest were after the “recall” period of 24–25 October . The gen2) was more common among female students compared to male students, whereas being overweight was more common among male students compared to female students. About one-fifth of students had a history of smoking, with more than one out of ten students reporting being active smokers. Of these active smokers, around 90% smoked traditional tobacco and between 0 and 10 cigarettes per day. Furthermore, working students  and non-commuters  smoked considerably more. Surprisingly, the percentage of occasional illicit drug users was similar to the percentage of active smokers, with men more involved than women . About 61% of the students were women, and the mean age of the sample was 22 years old. Being underweight (BMI ≤ 18.5 kg/mP = 0.001). Only one-third of students were aware of their lipid values, but awareness gradually increased to a peak of 60% during the 6th academic year . One-fifth of aware students  had total cholesterol values >155 mg/dL. Women more frequently had a lipid profile above normal levels .About three-quarters of medical students were aware of their blood pressure (BP), with an increasing awareness as the medical-study years progressed . Of these, only a minority,  had BP values higher than optimal ones (120/80 mmHg), and these were more frequently men .We measured the proportion of students having ideal cardiovascular health: only 22 students (1.9%) fell within this definition properly but, given the lack of some data regarding dyslipidemia and hypertension, it was estimated that the real presence in our population is 55 students (4.6%). This estimation was inferred through analyses made on the sample for which all the data were available . MoreoveFurthermore, we had the chance to evaluate this prevalence at different phases of students’ medical school career. Of interest, the number of subjects with high BP grew as the academic course years progressed, as well as the number of subjects consuming excessive amounts of alcohol and the number of students constantly stressed for university reasons B–D. In aP = 0.001) is alarming, although only 4% of them are severely underweight (BMI < 16 kg/m2). It is worth mentioning that, especially among the population below 40 years old, underweight persons have a 2.3-fold greater risk of CV disease [The main finding of this survey carried out in a large young Italian population, is a relatively high prevalence of traditional risk factors for atherosclerotic vascular disease in apparently healthy men and women in their third decade of life. Furthermore, these results were obtained from medical school students, in whom adequate knowledge of atherosclerosis mechanisms and strategies for prevention is expected. Of note, the CV risk profile worsened throughout the course of students’ medical school careers. Youth is a critical period: Not only is the correct awareness of individual risk exposure lacking , but it  disease .Beyond the low adherence to the Mediterranean diet, another interesting finding is the lack of insight regarding individual eating habits. In fact, even though 72% of students believed they adopted the Mediterranean diet correctly, only a quarter of them actually had a PREDIMED score >10. It is reasonable to expect similar data for salt intake: only 30% of students believed they use more salt than recommended while the literature reports that about 93% of young Italians exceed 5 g/day, a cut-off established by the World Health Organization (WHO) .Moreover, although medical students have an awareness of their BP and lipid profile greater than that reported in similarly aged subjects , it is sWe hypothesize that the psychological and time commitments that medical school has a role in this worsening trend. The university-related stress levels are alarming, with about 40% of the students being constantly stressed and 75% they have seen their stress level increase over the years. Such a psychophysical condition may predispose medical students to inappropriate behaviors such as high alcohol consumption, substance abuse, and smoking. Indeed, a recent study showed that stress levels among students have been associated with higher blood pressure . Not sur2, active smoking, hypertension, dyslipidemia, diabetes, CV family history, inadequate diet (PREDIMED ≤ 5), and absence of physical activity. This rate likely identifies with high specificity those subjects who are at high risk of future CV events. On the other hand, if in addition to the 8 main risk factors, minor risk factors are also considered, such as BMI ≤ 18.5 kg/m2 [Finally, the overall risk profile of this population of medical students is far from ideal, even though more than 6 out of 10 subjects believe that they adopt a healthy lifestyle. In fact, only a few students (4.6%) can be considered as having ideal cardiovascular health. Approximately 5% of students, regardless of gender, have at least 3 of the 8 main cardiovascular risk factors: BMI > 24.9 kg/m.5 kg/m2 , alcohol.5 kg/m2 , drug us.5 kg/m2 , sleep t.5 kg/m2 , up to 1Our study has some limitations that should be highlighted. As with all surveys, the ascertainment of lifestyle habits and medical history is subject to recall bias and to concerns of accuracy with self-reported data. Although a 44% response rate may not seem high, we consider it a satisfactory result. In fact, the survey was conducted as an easy-to-do initiative by medical students to medical students without any traditional academic support. The study indeed was not promoted by the university nor sponsored or publicized by any of the professors. We suggest that a peer-to-peer anonymous approach that we used enhanced the honesty and reliability of the responses, especially those regarding subjects’ personal spheres, such as lifestyle and habits. The actual response rate may be higher because it is possible that we did not notify all 2700 students formally enrolled in the medical school program given any probable absences from face-to-face lessons at the moment the flyer was distributed. Importantly, general characteristics, such as gender and age, of the non-responders are similar to those of the responders. Non-responders, in fact, being enrolled in the same university, are part of the same academic courses, the same city, and a similar social context.Our study is a cross-sectional description of different generations of students, so no clear conclusion can be made about the impact of medical education on each subject’s lifestyle and risk factors. We tried to mitigate this intrinsic limitation by including a specific section (fifth) of the questionnaire requiring students to self-evaluate the variations that they experienced during the course years. Only a prospective cohort study where medical students are evaluated at the beginning and at the end of their education may properly address this issue. Furthermore, by not having a control group, it is not possible to establish a causal link between the medical education path itself and the lifestyle changes and behaviors we observed among our students.The main strengths of this study include the large sample size and the innovative method of conducting the survey. We emphasize the practicality, convenience, and speed of data collection through the online questionnaire accessible via QR code. It may be that an eye-catching flyer with a “mysterious” QR code encouraged students, otherwise generally hesitant to complete surveys if not obliged, to fill in the questionnaire. To our knowledge, in the current medical literature there are only a few studies based on QR code surveys. We believe that this methodology can be widely used in the future to create reliable survey databases, especially in the COVID-19 era in which social interactions must be limited as much as possible . FurtherIn conclusion, the data from the UNIMI HEART survey remind us that the time has probably come to shift the gaze of primary prevention from adults to young people. Furthermore, the present study suggests that even a population considered “protected”, such as that of young medical students, can show an unsatisfactory cardiovascular risk profile, and that medical school itself, being demanding, long, and stressful, could have a role in worsening the lifestyle. To clarify this aspect, a prospective study is needed, and it would be desirable to conduct this study in other faculties . It seems like an appropriate time to question the medical education system, which trains its students without guaranteeing them the correct lifestyle it teaches. Such training issues also can affect patients: Can doctors in clinical practice properly educate their patients if they cannot apply the knowledge to themselves?


Oryctes rhinoceros, in Southeast Asia may clean up some of the persistent organic pollutants (POPs) for us if guarded in a controlled manner. Some xenobiotics persisting in our environment may cause harmful effects to the living creatures within their food web via a so-called “bioaccumulation effect”. The encounter of wild creatures with the POPs appears inevitable. Luckily, this study revealed that the proper breeding of the commonly seen beetle could degrade more than 95% of some studied POPs simply by ingestion. The beetle larvae tolerated different POPs at various extents, yet through an acclimation operation, the beetle’s mortality rate could be greatly reduced. Even though O. rhinoceros is considered a pest for some valuable corps, its removal of POPs in a natural, efficient and passive  manner makes this alternative promising and deserving of further explorations.A native, widely spread beetle, Oryctes rhinoceros larvae, a known pest of coconut trees in southeast Asia, and also the indicators of POP toxicity and the fate and degradability of the ingested POPs were assessed. The larvae were tested at various levels of the POPs and went through an acclimation process. Without acclimation, the tolerance limits of the larvae toward PCP, PAHs and DLN were 200, 100 and 0.1 mg/kg-soil, respectively, yet with acclimation, the tolerance levels increased to 800, 400 and 0.5 mg/kg-soil, respectively. Biodegradation rates of all the tested POPs were >90% by week 2, with <5% and nearly 0% remaining in the feces and body of the larvae, respectively. The results suggest that the use of the beetle larvae in soil POP decontamination is doable.The potential use of invertebrates as bioreactors to treat environmental pollutants is promising and of great interest. Three types of the persistent organic pollutants (POPs), namely pentachlorophenol (PCP), PAHs  and dieldrin (DLN), were spiked in soil and treated by using  Oryctes rhinoceros, were assessed. Furthermore, the persistent existence of POPs poses a bigger hazard to the environment. To date, many remedial methods have been employed to resolve soil POP problems through chemical or biological treatments [Long-term manufacturing and use of xenobiotic chemicals have resulted in the gradual accumulation of persistent organic pollutants (POPs) globally ,2,3,4,5.eatments ,14,15,16Oryctes rhinoceros is a well-known pest of coconut trees and also one of the most dominant beetle species in Southeast Asia. Its larval stage goes through three instars, and each takes roughly 2, 4 and 16 weeks, respectively, to complete. Due to its pest nature, insecticidal compounds, including 22-hydroxyhopane from Adiantum latifolium, have been used in its population control [O. rhinoceros have been reported: one was as the nutritional source of protein [O. rhinoceros, beneficial uses of its larvae and their potential applications and problems, refer to Shen et al. [ control . On the  protein  and the  protein . In eithn et al. .O. rhinoceros encounter each other, two major concerns of the POPs to beetle larvae were brought about immediately: one is the toxicity of the POPs and the other the beetle’s adaption to the POP-contaminated environment. Based on the life cycle of the larvae, a long, nearly six months of the larval period gives them more time to adapt to toxin consumption and also allows them to ingest/degrade quite a great deal of the pollutants. The extents of ingestion and/or digestion of the POPs by the larvae have not yet been studied and were worthy of further investigation. To assess the adversary effects of the POPs, a mimicked larval breeding environment was constructed. Several designated incubators were set up with fixed amounts of substrate and various types of POP-contaminated soils. Five indicators: the larval death rate, growth rate, cumulative feces production, substrate conversion ratio and percent of the POP degradation, were monitored weekly over the entire testing period. By doing so, the toxicity, the larval adaption and the degradability of the POPs were to be accessed. Mass flow of each studied POP was also traced to confirm the biodegradability of the tested POP.When the POPs and the Upon the completion of this study, the extent of the POP’s impact, the ability of larval adaptation to the POPs and the degradability of the POPs by the beetle larvae were to be fully understood. This information is deemed as important data on the management of the POP-contaminated sites, on the evaluation of the larval survival and the further applications of the POP-contaminated soil cleanups. Although the use of rhinoceros beetle larvae as bioreactors in treating environmentally persistent pollutants has not yet been demonstrated. Use biological treatment alternatives are commonly regarded as environmentally friendly and cost-effective alternatives. The use of large insects as bioreactors on pollutant removal via proper site management operations looks promising.The tested larvae were captured from their wild habitats, mainly from decayed wood stumps, and bred using a commercially available larval substrate, namely the fermented product of dust wood, leaves and soil. For information regarding an overall larva’s handling process and life span, please refer to Shen et al. . The spiA 37.5 × 23 × 15 (length × width × height in centimeters) rectangular plastic box  served as the larval incubator. Several small holes were drilled on 2 sides of the incubator for ventilation. At time zero, each incubator was filled with 1.5 kg of the soil and substrate mixture with the designated toxin content, which was maintained at ambient room temperature and 45% moisture content during the experimental period. In approximately 2 months, 2 runs were conducted in a series. Run 1, the toxicity test, was conducted for 4 weeks for various PCP, PAH, and DLN contents, which were topped at 200, 800 and 2 mg/kg, respectively. The samples of the soil/substrate mixture and feed were collected every week and the residual POP amount was quantified. Run 2, the acclimation test, was conducted for another 4 weeks to confirm the effect of acclimation on the beetle larvae. The larvae, which experienced the POPs and survived in Run 1, were considered as acclimated ones. The mortality rates of the acclimated and unacclimated larvae were examined, and the weekly residual levels and fate of each POP during the experimental period were determined.For the toxicity test , 14 incubators were set up. Each incubator was filled with a 1.5-kg mixture of soil and substrate at a 1:1 ratio and 20 larvae. Among these 14 incubators, 2 were free of toxins and served as blanks, 3 contained PCP levels of 50, 100 and 200 mg/kg-soil, 5 contained PAH levels of 50, 100, 200, 400 and 800 mg/kg-soil and 4 contained DLN levels of 0.1, 0.5, 1.0 and 2.0 mg/kg-soil. In the acclimation and POP degradation test , 14 incubators were set up, among which 2 were free of toxins and served as blanks, 4 contained PCP levels of 200 and 800 mg/kg-soil, 4 contained PAH levels of 100 and 400 mg/kg-soil and 4 contained DLN levels of 0.1 and 0.5 mg/kg-soil. In other words, two incubators each received 15 either acclimated or unacclimated larvae in each POP level were arranged so that whether the toxin-exposed larvae survived exposure to a higher level of toxins could be tested. The acclimated larvae were the surviving larvae from the toxicity test that underwent exposure levels of 200, 50 and 0.1 mg/kg-soil of PCP, PAHs and DLN, respectively, for 4 weeks.During each weekly sampling of the incubator operation, the incubated larvae were removed from the incubator and weighed, and the average weight of the larvae was determined to minimize disturbance. Subsequently, the larval feces (approximately 8–10 mm in size) was screened and separated from the substrate by using a 4-mm sieve. Finally, both the feces and substrate were oven-dried at 105 °C to determine their mass on a dry weight basis. By using the obtained data, the following 4 POP toxicity indicators were determined:To determine the POP amount, an additional 2 g of the soil/substrate mixture or feces were randomly sampled from the collected sample through extraction using 10 mL of a mixture of acetone and hexane at a 1:1 ratio (by volume) and sonicated using a sonicator (Misonix 3000). Subsequently, the sample was centrifuged at 10,000 rpm using a centrifuge , and the centrate was analyzed using gas chromatography–flame ionization detector  equipped with a PET-5 capillary column from Sigma-Aldrich, Inc. The oven temperature was initially 50 °C for 5 min, then increased to 300 °C at a rate of 10 °C/min and then maintained at 300 °C for another 15 min before program termination. A fixed amount of the internal standard 2,3,4-tribromophenol was added to the centrate to calibrate analytical errors that may have occurred during the GC-FID analysis. For further information on sample extraction, analysis, and quantification, refer to Chen et al. .To determine the weekly removal rate of POPs, the net difference between POP intake and excretion was calculated, and the resulting value was divided by the total intake (Equation (5)). To trace the distribution of each POP during Run 2, total POP intake and excretion, the POP amount in larval bodies and POP amount in gut contents were determined for each larva to assess the percent distribution of POPs in each portion of the specimen. The percent degradation of POPs was calculated using Equation (6).For data quality assurance and quality control, all measurements were performed in triplicate and presented as averaged values. Percentage error (% error) of each measurement was calculated by dividing the standard deviation by the mean of the measurements, and a 5% limit was adopted to ensure data consistency. As for experimental duplications, at least one duplicated incubator was duplicated in Runs 1 and 2, and average values were calculated and reported. Yet, for the clarity of the plotted figures, the standard deviation of the data was not plotted.For the results of the toxicity test (Run 1), As for the results of the acclimation and POP degradation test (Run 2), though the four POP toxicity indicators were also examined; however, due to their similar trends as concluded in the toxicity test, these data, except for the mortality rates, are not shown here. Thus, the larval mortality rates and weekly degradation rates of the spiked POPs are given in The fates of POPs were further studied by determining the amount of POP in larval feces, bodies and gut contents. In PCP incubators, 1.6% residual PCP was found in the feces and a total of 0.3% was observed in larval bodies and gut contents, which indicated that 98.1% of PCP was degraded through larval ingestion a. In PAHOryctes rhinoceros encountering POPs are limited. Results of this study revealed that the impacts of POP dosages on the larval growth indicators looked obvious and indisputable. Once a higher and higher amount of POP was ingested, larvae’s mortality became the final resolution of such an encounter. However, based on the obtained results, the use of beetle larvae in environmental toxin removal appeared promising in three observations: (1) the larval survival rates and POP tolerance levels increased if larvae properly acclimated, which revealed the potential elimination of POPs in the fields through certain site management, (2) the four POP toxicity indicators might serve as operational monitoring items during the field remedial treatment, and finally (3) the complete degradation of the POPs makes the larval remediation a natural and environmentally friendly alternative.Up to date, studies directly related to Oryctes rhinoceros as a bioreactor to treat total petroleum hydrocarbons (TPHs). Their results revealed that with acclimation, beetle larvae could stand high doses of TPHs and resulted in a higher TPH removal efficiency over time. This implied the potential uses of the beetle larvae as bioreactors in treating various pollutants.The potential use of beetle larvae to clean up our environment sounds non-realistic. Yet, some characteristics of invertebrates, such as their long-term inhabitation in soil, capacity to chemically adapt, their high production rate and their global presence, make them excellent candidates in soil decontamination and this kind of decontamination might have happened without our notice. As a similarity, the earthworms have been widely studied for soil pollution treatment ,25,26,27O. rhinoceros is a pest in nature. Certain risks exist if used in soil decontamination. To fully adopt the larval bioreactors in pollutant decontamination, both human health and ecological risk assessments are of concern. For detailed risk assessment information, refer to the USEPA [Even the obtained results look encouraging, yet some precautions deserve further considerations. Firstly, he USEPA , and forhe USEPA . From anhttps://www.epa.gov/risk, accessed on 31 August 2021) that fully explains the contents of human health and/or ecological risk assessments, and the other by Li [Two references were added in the last paragraph of the “Results and Discussion” section regarding the risk assessment issue. One was the USEPA risk assessment website  without exposure to POPs, the tolerance levels of larvae toward PCP, PAHs and DLN were 200, 100 and 0.1 mg/kg-soil, respectively. At or below such POP levels, the larvae exhibited <20% mortality and could be properly acclimated, (2) when the acclimated larvae encountered 800, 400 and 0.5 mg/kg-soil levels of PCP, PAHs and DLN, respectively, their mortality was minimal and >90% POPs were degraded by week 2. By contrast, at the same POP levels, most unacclimated larvae died. Appropriate acclimation of the larvae could stabilize the effects of real POP-contaminated field treatment, (3) the results of a fate analysis revealed that >98% and >95% of PCP and PAHs, respectively, were degraded through the larval intake, with a minimal POP amount remaining in larval feces and bodies, and (4) by contrast, only approximately 35% of DLN was degraded through larval ingestion. Nearly 58%, 6.4% and 0.8% of spiked DLN were accumulated in larval gut contents, bodies and feces, which indicated a large accumulation of DLN in the larvae.Based on these results, the degradation of certain POPs in the field might have already happened all the time. Yet through the uncovered data, the impacts of POPs to beetle larvae were revealed, proper site management via the proposed indicators learned and the potential of better performance on POP-contaminated soil remediation proposed. More studies at this point might help the larvae already there to recover our environment in a safer and faster way.


Type 2 diabetes mellitus (T2DM) is a global pandemic with a significant negative impact on health-related quality of life. Worldwide, the prevalence of T2DM has almost doubled since 1980. Although multiple systematic reviews and clinical trials have suggested that electroacupuncture could be effective for T2DM treatment, whether it can improve glucose and lipid metabolism has not been systematically reviewed.We searched PubMed, Medline, Embase, Web of Science, Cochrane Central Register of Controlled Trials, China National Knowledge Infrastructure, Chinese Biomedical Literature Database, Chinese Scientific Journal Database, and Wan-Fang Database from the date of creation to December 2022. Language is limited to both Chinese and English languages. Clinical randomized controlled trials related to acupuncture for T2DM were included in this study. Fasting plasma glucose, fasting insulin, lipid profile, and glycated hemoglobin levels were the primary outcomes. In addition, we manually retrieved other resources, including reference lists of identified publications, conference articles, and gray literature. Research selection, data extraction, and research quality assessments were independently completed by 2 researchers.This study provides more options for clinicians and patients to treat obese patients with type 2 diabetes.In this study, we aimed to summarize and assess the effectiveness and safety of EA as a supplemental method to treat T2DM patients from clinical trials and provide more options for clinicians and patients to treat T2DM.https://inplasy.com/inplasy-2021-8-0008/).This study was registered at INPLASY with registration number INPLASY202180008 (INPLASY202180008. In China alone, the prevalence of diabetes has increased tenfold in 30 years, from 0.67% in 1980 to 10.4% in 2013. From 2015 to 2017, the epidemiology of thyroid, iodine nutritional status, and diabetes mellitus in 31 provinces of China conducted by the endocrinology branch of the Chinese Medical Association showed that the prevalence of diabetes in people aged 18 and above was 11.2%. With the development of science and technology, people's understanding of T2DM has gradually deepened, and the detection and treatment methods for T2DM are gradually increasing. In addition to a variety of therapeutic drugs and metabolic surgery for obese T2DM patients, traditional Chinese medicine has also gradually played a role in the treatment of T2DM.Type 2 diabetes mellitus (T2DM) is characterized by hyperglycemia, a relative lack of insulin and insulin resistance, and is a chronic metabolic disease. The incidence rate of T2DM is increasing dramatically worldwide. The accompanying hyperglycemic state will cause abnormal metabolism of the patients’ metabolism, not only cause diabetic nephropathy and other related complications, but also increase the coronary atherosclerosis significantly, and the incidence of coronary atherosclerosis is increased significantly in 2 cases. The prevalence of cardiovascular and cerebrovascular diseases such as myocardial infarction.–6Acupuncture, the most distinctive external therapy in traditional Chinese medicine, has long been applied to assist in the control of blood glucose and lipid levels in T2DM patients. Electroacupuncture is a perfect combination of modern science, technology, and acupuncture therapy. Animal experimental studies have shown that electroacupuncture at different frequencies can activate cholinergic nerves and stimulate the release of β-endorphin and other endogenous opioid peptides by the adrenal glands, thus promoting the secretion of insulin.Although multiple systematic reviews and clinical trials have suggested that electroacupuncture could be effective for T2DM treatment, whether it can improve glucose and lipid metabolism has not been systematically reviewed. This meta-analysis aimed to summarize and assess the effectiveness and safety of EA as a supplemental method to treatT2DM patients from clinical trials and provide more options for clinicians and patients to treat T2DM.2 This protocol was registered on the International Platform of Registered Systematic Review and Meta-analysis Protocols (INPLASY202180008).The systematic review was performed following the guidelines of the Preferred Reporting Items for Preferred Reporting Items for Systematic Review and Meta-Analysis Protocols 2015.2.12.1.1The type of literature gathered must be based on random clinical research (RCT) trials. Non-RCTs will be ruled out, including studies such as case reports and studies without sufficient information about the randomized method or process. Do not limit the use of blind methods, but the authors should clearly claim that they performed random grouping.2.1.2The diagnosis of T2DM was based on the diagnostic criteria for diabetes in 1999. Meanwhile, the participants who were included had no limitations in age, sex, region, citizenship, and nationality. Cases related to serious diseases, pregnancy, and drug-induced T2DM were excluded.2.1.3One therapeutic intervention used in the experimental group included EA. Therefore, acupoint catgut embedding, cupping, acupoint injection, blood-letting therapy, and herbal medicine should be excluded.2.1.4The primary outcome measures will be fasting plasma glucose, glycated hemoglobin, fasting insulin, and lipid profile. In addition. Weight loss (kg), homeostatic model assessment of insulin resistance level, C-reactive protein level, dropout, side effects, and rebound will be assessed.2.2We electronically searched PubMed, Embase, Web of Science, the Cochrane Central Register of Controlled Trials, China National Knowledge Infrastructure, Chinese Biomedical Literature Database, Chinese Scientific Journal Database, and Wan-Fang Database from the date of creation to December 2022. In addition, we manually retrieved other resources, including reference lists of identified publications, conference articles, and gray literature.2.3We constructed a search strategy using a combination of medical subject heading terms and text words. We will verify the systematic review and meta-analysis that also focused on our topic and the bibliographies of eligible studies in order to add any additional eligible studies. For example, we summarized the search strategy for PubMed Table .2.4Studies will be identified using NoteExpress 3.2.0. After the initial removal of duplicate studies, 2 reviewers independently screened the titles and abstracts based on the eligibility criteria. Full-text studies will be retrieved for all potentially includable SR or SR protocols. If studies contain insufficient information to make a decision about eligibility, they will try to contact authors of the original reports to obtain further details. The process of identification and selection of literature is shown in Figure 2.5I2 test was used for the statistical analyses. When I2 was <50% or P > .05, there was no statistical heterogeneity between the studies, and the fixed-effect model was selected to combine the effect amount; otherwise, the random-effects model was adopted.We will use Review Manager 5.4 software provided by the Cochrane Collaborative Network for statistical analysis. For continuous variables, the mean and SD of each study were obtained and pooled as the mean difference or standardized mean difference with a 95% confidence interval. Statistical heterogeneity analysis was performed for the included RCTs. The Cochrane 2.6Q test and I2 statistics. According to the Cochrane Handbook, the thresholds for the interpretation of the I2 statistics are as follows: 0% to 40%, might not be important; 30% to 60%, may reflect moderate heterogeneity; 50% to 90%, may indicate substantial heterogeneity; 75% to 100%, represents considerable heterogeneity After statistical heterogeneity is established, the researchers will search for possible sources from the clinical and methodological perspectives and perform subgroup or sensitivity analysis to detect the possible causes of heterogeneity.Statistical heterogeneity among the studies was assessed using the 2.7We used funnel charts to assess reporting biases. When a sufficient number of included studies  are available, we will conduct a test for funnel plot asymmetry using the Egger method.2.8To test the robustness of the main decisions in the review process, we conducted a sensitivity analysis. The main analysis points included the impact of method quality, sample size, and missing data on the study. The meta-analysis will be reused, and more inferior-quality studies will be excluded. The results are compared and discussed based on the results.2.9The quality of systematic reviews will be evaluated using the grading of recommendations, assessment, development, and evaluation. Five downgrading factors, including risk of bias, inconsistency, indirectness, imprecision, and publication bias, were assessed. The assessment results were divided into 4 levels: high, moderate, low, or very low.3 With the arrival of the aging era, T2DM has posed a serious burden to many families and society. As a metabolic disease characterized by hyperglycemia, with the continuous elevation of blood glucose, the body will develop from normal to prediabetes, and then develop into diabetes and complications. In this process, any intervention measures such as improving life, diet, and exercise habits can improve the pathological state leading to the increase of blood glucose–13 , help the body slow down the increase of blood glucose, and even reverse the hyperglycemia to a lower level.In recent years, with the continuous accumulation of evidence-based medical evidence for T2DM and the change of disease spectrum, people's understanding of T2DM has gradually changed.However, the current treatment for T2DM can only be hypoglycemic drugs for the direct cause, but long-term drug use has also influenced people's lives and work. In addition, many complications caused by poorly controlled diabetes have a significant impact on quality of life. Meanwhile, more and more patients began to realize the single limitation of controlling diseases only by hypoglycemic drugs, so they sought the help of traditional Chinese medicine from the hospital of traditional Chinese medicine. Therefore, how to use traditional Chinese medicine to prevent and improve the symptoms of T2DM interventions and find an intervention method that can improve insulin resistance and reduce side effects in patients with high glucose and high fat T2DM has become the main research direction of T2DM, and is also one of the focuses of traditional Chinese medicine in the field of diabetes research. In this study, we aimed to summarize and assess the effectiveness and safety of EA as a supplemental method to treat T2DM patients from clinical trials and provide more options for clinicians and patients to treat T2DM.Conceptualization: Yiran Han, Xiaochao Gang.Data curation: Yiran Han, Zeyuan Lu.Formal analysis: Shaotao Chen, Meng Meng.Methodology: Zeyuan Lu, Ting Pan.Software: Yiran Han, Tianjiao Gao.Supervision: Mingjun Liu.Writing – original draft: Yiran Han, Xiaochao Gang.Writing – review & editing: Zeyuan Lu, Mingjun Liu.