ADAM12 gene expression and hepatocellular carcinoma (HCC), the prognostic value of this relationship, and the potential mechanisms influencing HCC development were evaluated. The results showed that the ADAM12 gene was significantly and highly expressed in liver cancer tissue. The high expression of the ADAM12 gene in liver cancer tissue significantly and positively correlated with T stage, pathological stage, and residual tumour. Kaplan–Meier and Cox regression analyses revealed that ADAM12 gene expression is an independent risk factor influencing the prognosis of patients with liver cancer. Pathway analyses of ADAM12 in HCC revealed ADAM12-correlated signalling pathways, and the expression level of ADAM12 was associated with immune cell infiltration. In vitro experiments demonstrated that the expression level of ADAM12 in Huh-7 and Hep3B cells was significantly higher than that in other HCC cells. ShRNA transfection experiments confirmed that the expression levels of TGF-β and Notch pathway-related proteins were significantly decreased. An EdU cell proliferation assay showed that a low level of ADAM12 gene expression significantly inhibited the proliferative activity of HCC cells. Cell cycle experiments showed that low ADAM12 expression blocked the G1/S phase transition. Overall, this research revealed that high ADAM12 gene expression implies a poor prognosis for patients with primary liver cancer. In addition, it is a potential indicator for the diagnosis of liver cancer.Disintegrin and metalloproteinase 12 (ADAM12) is thought to trigger the occurrence and development of numerous tumours, including colorectal, breast, and pancreatic cancers. On the basis of The Cancer Genome Atlas (TCGA) datasets, in this study, the relationship between  According to 2018 global cancer statistics, liver cancer is the 4th leading cause of cancer death3. Liver cancer has no significant clinical symptoms during the early stages; it is often diagnosed in the intermediate or late stage, at which point, it is highly malignant. The recurrence rate is high. In the past, conventional or single treatment often increased recurrence risk due to residual liver tissue and poor patient tolerance of chemoradiotherapy5. In recent years, emerging therapeutic methods, including molecular targeted therapeutic drugs and immunity-inducing drugs, have displayed better therapeutic effects and weaker side effects in advanced hepatocellular carcinoma than traditional therapies7.Liver cancer is a complex disorder and one of the most prevalent malignant tumours in the digestive system. It is caused by multiple factors, and the primary pathological type is hepatocellular carcinoma (HCC)2+-dependent metalloproteinase enzymes10. To date, as many as 40 members of the ADAM protein family have been identified, and they have been shown to regulate cell phenotypes through their effects on cell adhesion, migration, proteolysis, and signal transduction12. In recent years, several studies have shown that this family promotes the occurrence and development of tumours by targeting cell development, regulating the inflammatory response, and releasing membrane-bound proteins14.Disintegrin and metalloproteinases (ADAMs) are transmembrane glycoproteins anchored to the cell membrane that belong to the Metzincin superfamily of ZnADAM12 gene is generated by ADAM splicing; is primarily found in the bone and cartilage; and is expressed in the brain, liver, heart, and muscles17. The human ADAM12 gene is located on chromosome 10q26.3, and in tumour cell lines, it is expressed in two different spliced forms16. Additionally, several studies have shown that the expression of the ADAM12 gene is upregulated in pancreatic, colorectal, gastric, lung, and breast cancers and leads to poor prognosis21. Notably, the ADAM12 gene is a binding partner of the activated protease C receptor during liver fibrosis, and both ADAM12 and protease C are highly expressed in liver cancer and liver fibrosis22. Liver fibrosis and cirrhosis caused by chronic hepatitis B are important risk factors for liver cancer24; therefore, ADAM12 is closely related to liver injury and liver cancer. Nonetheless, reports on ADAM12 in primary liver cancer remain rare. Although studies have suggested that the ADAM12 gene may be related to the invasion and progression of liver cancer cells25, the potential mechanisms and clinical relevance of these actions remain to be clarified.The ADAM12 gene expression to the prognosis of liver cancer using liver cancer samples obtained from the TCGA database. Moreover, we clarified the biological pathway and mechanism of the ADAM12 gene implicated in the regulation of the occurrence and development of liver cancer via gene enrichment analysis (GSEA) and immune cell invasion analysis. Our findings will help in the discovery of novel prognostic biomarkers and prediction of the potential molecular mechanisms influencing the prognosis of liver cancer.In this study, we evaluated the contribution of ADAM12 gene expression level. Comprehensive information on these clinical data is provided in Table The clinical data used to characterize 374 patients with hepatocellular carcinoma were downloaded from the TCGA database and divided into high and low groups based on the median ADAM12 gene mRNA in different tumour tissues and normal tissues were analysed by Wilcoxon signed-rank test  and cholangiocarcinoma (CHOL), significant differences were noted between tumour tissue and normal tissue. In hepatocellular carcinoma, the expression of the ADAM12 gene in tumour tissues was significantly higher than that in normal tissues (p = 6.4e−06)  , age (p = 0.03), sex (p = 0.02), pathological stage (p = 7.2e−03), and histological grade (p = 0.01)     Fig. A. The me05) Fig. B–D. A un1) Table . A multi26) Fig. E.Figure ADAM12 gene effects in liver cancer, data from the TCGA database were utilized to perform gene set enrichment analysis (GSEA). A Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of ADAM12 gene expression samples was performed on the basis of MSigDB enrichment data . On the basis of P value, NES value, and FDR value criteria, significantly enriched pathways, including the Notch, GnRH, Hedgehog, TGF-β, JAK/STAT, MAPK, Calcium, Neurotrophin, and Fc epsilon signalling pathways, were found to be enriched in the group with high ADAM12 gene expression , immature dendritic cells (p < 0.01) and follicular T cells (p < 0.01), but negatively correlated with helper T cells 17 (p < 0.01)  and Spearman correlation to analyse the association between ADAM12 expression levels and subsets of infiltrating immune cells. The expression of the 01) Fig. . In addi01) Fig. .Figure 5p < 0.05). Next, we detected the protein expression level of ADAM12 in different HCC cell lines. A Western blot analysis indicated that the ADAM12 protein expression level was relatively high in Huh-7 cells and Hep3B cells . In addition, we validated shRNA interference sequences. The results showed that the third interference sequence tested was effective in inhibiting ADAM12 expression .To verify the bioinformatics analysis results and determine the correlation between ADAM12 gene expression and liver cancer, we first compared the expression of ADAM12 in cancer tissues and adjacent tissues taken from liver cancer patients. The results showed that the expression level of the ADAM12 protein in the cancer tissues was significantly higher than that in the adjacent tissues 25. Moreover, Nathalie's team proposed that RACK1 is a chaperone for ADAM12 promotion of liver fibrosis22. Therefore, the effect of ADAM12 expression on hepatic fibrosis implies that ADAM12 might be closely related to the occurrence and development of liver cancer.Primary liver cancer is caused by various complex factors; however, it primarily results from increased liver cell damage. Liver fibrosis is a chronic pathological process caused by excessive deposition of ECM, which induces liver injury and subsequent changes in the microenvironment to induce primary liver cancer occurrence and developmentADAM12 gene in primary liver cancer tissues was significantly higher than that in paracancerous tissues. The survival analysis and logistic regression analysis revealed that patients with high ADAM12 gene expression exhibited a low survival rate and poor prognosis. The multivariate Cox regression analysis showed that the ADAM12 gene is an independent risk factor for HCC. Tumour staging (TNM) is used to evaluate the number and location of malignant tumours in vivo, and it is used to guide clinical treatment, to a certain extent, since each stage (I to IV) is associated with different prognostic characteristics34. In the present study, the expression level of the ADAM12 gene was significantly and positively correlated with tumour size and pathological stage, indicating that the ADAM12 gene can potentially be used as an indicator of liver cancer stage.We analysed the integrated data and discovered that the expression level of the 37. We found that highly expressed ADAM12 was primarily enriched in the Notch, GnRH, Hedgehog, TGF-β, JAK/STAT, MAPK, Calcium, Neurotrophin, and Fc Epsilon signalling pathways. Previous studies have implicated Notch in the proliferation, migration, and metastasis of various cancer cells40. However, Notch signalling may play a contradictory role due to the interaction between pathway regulatory mechanisms and the microenvironment42. However, several lines of evidence have indicated that Notch signalling pathways are significantly associated with cirrhosis, liver fibrosis and HCC45. Similar to the Notch pathway, the TGF-β signalling pathway plays a dual role in the development of HCC and inhibits the early development of HCC46. Nonetheless, hepatoma carcinoma cells undergo the EMT after responding to TGF-β, and therefore, the migration and invasion of liver cancer cells are increased48. Importantly, our study validated the association of ADAM12 gene expression with the Notch and TGF-β signalling pathways. Viral hepatitis and NASH are important causes of liver cancer development. Liver fibrosis due to viral hepatitis and NASH has been shown to be a risk factor for the development of hepatocellular carcinoma51. It has been suggested that ADAM12 is significantly associated with liver fibrosis52. The results of this study showed that ADAM12 was positively correlated with the expression of TGF-β (a marker of fibrosis) in liver cancer. In addition, activation of the Notch pathway increases the rate of liver fibrosis53. Therefore, the expression of the ADAM12 gene in the liver may promote the progression of hepatocellular carcinoma through the formation of liver fibrosis, but the data are not conclusive. In contrast to the complex roles of the Notch and TGF-β signalling pathways, the Hedgehog signalling pathway has been found to be consistently abnormally activated in association with tumour progression, metastasis, and drug resistance56. In HCC, the high expression of SMO and GLI1, members of the Hedgehog signalling pathway, directly triggers the formation of larger tumours and is significantly associated with recurrence58. In addition, the JAK/STAT and MAPK signalling pathways are associated with antitumour immunity59. Both the JAK/STAT and MAPK pathways have been confirmed to be crucial targets in related studies of HCC resistance61.Excessive cell proliferation and rapid metastasis are typical features of the sustained development of malignant tumours; these processes are regulated by multiple signalling pathways in vivo62. Notably, by upregulating IL-10 expression, HIG2 facilitates HCC evade the killing induced by NK cells, promoting HCC cell recurrence and metastasis63. Because of these outcomes, immune cell therapy in hepatocellular carcinoma has broad application prospects. The expression of ADAM12 may be associated with immune cell infiltration in hepatocellular carcinoma; these invading cells include macrophages, immature dendritic cells, follicular T cells, and helper T cells. These findings may provide potential insights into the use of immunotherapy for patients with liver cancer.In recent years, tumour immune evasion has been a hot topic in research related to tumour treatment. Several immune cells, including macrophages, T cells, and autonomous NK cells reside in the microenvironment of tumour tissues. These cells directly or indirectly affect the tumour cell microenvironment and regulate tumour cell behaviour. For instance, in hepatocellular carcinoma, the antigen-specificity of invasive T cells is highly correlated with tumour control that is specifically manifested as CD8 + T cells, which are associated with an effective antitumour responseADAM12 gene expression and hepatocellular carcinoma. In addition, we evaluated the feasibility of ADAM12 gene expression as a prognostic factor for hepatocellular carcinoma and predicted the possible ways that ADAM12 affects the progression of liver cancer, thereby providing a reliable basis for the prevention and prognosis of hepatocellular carcinoma. Finally, inhibition of ADAM12 gene expression significantly blocked the G1 phase transition and thus reduced cell proliferation in our experiments. Furthermore, knocking down ADAM12 expression inhibited Notch pathway activation and reduced the TGF-β expression level. Although Huh-7 and Hep3B cell lines were mainly used in the experiments, we will verify the results with human and murine HCC tissues in future studies. In summary, we speculate that ADAM12 exhibits great potential for promoting HCC progression, leading to a poor prognosis for patients with HCC.In conclusion, we used bioinformatics to explore the close correlation between high https://portal.gdc.cancer.gov/repository), which is a publicly available database. The expression information of the ADAM12 gene was obtained through high-throughput sequencing of TCGA data. First, an analysis of differential ADAM12 gene expression was performed, and the results are presented with a box diagram and paired difference diagram. Ultimately, the RNA-Seq HTSeq-Counts gene expression data for liver cancer patients and the clinical data were analysed with R software (version 3.6.3).The gene expression data and basic clinical characteristic data, based on 50 samples of normal liver tissue and 374 samples of liver cancer tissue , were downloaded from the TCGA website (https://www.gsea-msigdb.org/gsea/index.jsp). Based on the median ADAM12 gene expression level, the patient data were categorized into high and low groups. Then, these TCGA data were prepared in text format and imported into GSEA software. Notably, the gene set arrangements were repeated 1,000 times for each analysis. For the GSEA, the P value, FDR value, ES value, and NES value were mainly assessed; these are widely recognized screening parameters. Gene enrichment sequencing was first performed based on the NES values; then, in the gene enrichment analysis, the genes with expression differences that met the p < 0.05 and FDR < 25% criteria were considered to be significantly differentially expressed64.GSEA software was downloaded from the GSEA website  was used to analyse the prognosis of 25 types of tumours. The ADAM12 gene in hepatocellular carcinoma was analysed using this website, and overall survival was estimated by calculating logarithmic grade P values and hazard ratios (HRs).The Kaplan–Meier Plotter ; the SMCC7721 cells were cultured in RPMI-1640 medium . All the cells were cultured in the suitable medium containing 10% foetal bovine serum  at 37 °C with 5% COADAM12 shRNA interference (pRNAT-U6.1/Neo) and negative plasmids were produced by Ruibo Biological Technology Co., Ltd. . Cells were transfected with short hairpin RNA (shRNA) with the following sequence: 5′-GGGTTCACGAGTGTGCAAT-3′ against ADAM12. Briefly, cells were plated into 12-well cell culture plates. When the cells reached 60%-80% confluence, transfection was carried out using Lipofectamine® 3000 Reagent  according to the manufacturer's instructions. The transfection efficiency was detected by Western blotting. ImageJ version 1.52 software  was used for the densitometry analysis.The proliferation ability of cells was detected with an EdU assay kit following the protocol provided by the manufacturer. In brief, cells were seeded into 24-well plates. After adherence, the cells were incubated with EdU solution for 2 h. Following three washes with PBS, the cells were fixed with 4% paraformaldehyde, neutralized with glycine, and reacted with Apollo® fluorescence dyes. Finally, Hoechst 33,342 reaction solution was used to stain nuclei. The cells were observed by inverted fluorescence microscopy  at 100 × magnification. The proportion of EdU-positive cells (red fluorescence) to Hoechst-stained cells (blue fluorescence) was calculated. The results were analysed using ImageJ version 1.52 software.The cells were dissolved in RIPA lysis buffer and PMSF , and cell lysates were collected and then centrifuged for 10 min at 12,500 g. The protein concentration was measured with an ND2000 microspectrophotometer . Proteins were separated by SDS–PAGE (10% gels), transferred to PVDF membranes, blocked with 5% free fat milk at room temperature for 2 h and incubated with primary antibody overnight in a 4° refrigerator. The next day, the membranes were incubated with the corresponding secondary antibody at room temperature for 2 h. ECL (chemiluminescence) fluid and an imaging system were used for exposure and data analysis. Protein expression levels were semiquantified by ImageJ version 1.52 software. The following primary antibodies were used: anti-ADAM12 , anti-ACTIN (US Sigma A1978 1:5000), anti-Jagged1 (US CST/2620S 1:1000), anti-Notch2 , anti-Hes1 (US CST/11988S 1:1000) and anti-TGF-β .Eight pairs of clinical liver cancer samples were collected, and paraffin sections were obtained from the Department of Pathology, Yijishan Hospital, Wannan Medical College. The expression of ADAM12 in the liver cancer tissues and adjacent tissues was detected by IHC. First, xylene was used to deparaffinize and a diluted alcohol gradient was used for hydration. Tissue sections were treated for antigen retrieval with citrate antigen retrieval buffer. Then, the tissue was placed in 3% hydrogen peroxide to block endogenous peroxidase and blocked with serum for 30 min. The tissue was incubated with anti-ADAM12 antibody overnight at 4 °C. The next day, labelling with the corresponding secondary antibody was performed, followed by DAB colour development. Finally, the stained tissue was visualized with a light microscope.Cells were collected according to kit instructions  and immobilized overnight with a final concentration of 70% ethanol. Next, the cells were washed three times with PBS and centrifuged to remove the supernatant. Finally, the cells were stained according to the instructions of a cell cycle assay kit and maintained in the dark for 30 min at room temperature. A flow cytometer  was used to detect the cell cycle distribution.ADAM12 gene expression between the normal liver tissue and liver cancer tissue. In addition, Wilcoxon signed-rank test, logistic regression, and Kruskal–Wallis tests were performed to analyse the relationship between ADAM12 gene expression and clinicopathological features, excluding patients with incomplete clinical data. The overall survival curve was plotted on the basis of a Kaplan–Meier analysis, and univariate and multivariate Cox regression analyses were performed to assess the effects of the ADAM12 gene and clinical characteristics on overall survival. Experiments were performed in triplicate, and the data are shown as the means ± SD. The statistical significance of differences was determined by Student's t test for comparisons of two groups. p < 0.05 was considered to be statistically significant.R software (version Rx64 V3.6.3) was used for statistical analysis. Wilcoxon rank-sum test was performed to analyse the difference in the This study was approved by the Ethics Committee of Scientific Research and New Technology of Yijishan Hospital of Wannan Medical College with the approval number of 202095, and obtained informed consent from all patients who participated in this study. All methods were performed in accordance with the Helsinki declaration guidelines and regulations.We agree to publish the manuscript.Supplementary Information 1.Supplementary Information 2.


Blaps rynchopetera Fairmaire.To explore the potential immunomodulatory effects of total extract and different polar parts from Phagocytic activity was evaluated by neutral red assay, and the effect of the immune function was investigated by normal and immunocompromised mice models.In vitro, total extract, as well as chloroform, ethyl acetate, n-butanol and water fractions could individually enhance the phagocytic ability of mouse peritoneal macrophages; in addition, chloroform and ethyl acetate fractions had an increasing tendency when combined stimulation with lipopolysaccharide (LPS). In vivo, ethyl acetate fraction (EAF) could enhance the immune organ index, increase the serum hemolysin level and peripheral blood immune cells of immunocompromised mice, while for normal mice, the effect was inconspicuous.Blaps rynchopetera extracts had noteworthy immunomodulatory effect, especially for individuals with immune disorders. Studies have revealed that cancer and autoimmunity are closely relatedBlaps rynchopetera Fairmaire is one of the medicinal insects that belongs to the family Coleoptera Heteromera (Coleoptera) of beetles. It is also known as the smelly fart bug or stink beetle due to its smelly defensive secretion; it is used to treat fever, cough, gastritis, whitlow boils, tumors and other diseases,,Insects not only have nutritional value but also have medicinal valueB. rynchopetera could individually increase the phagocytosis index of mouse macrophages to varying degrees. Ethyl acetate fraction (EAF) boasts superior activity and can significantly relieve cyclophosphamide-induced immune deficiency in mice, increase the production levels of the hemolysin antibody, raise the level of immune cells in peripheral blood, and increase the spleen and thymus indexes. Results indicate that this fraction has a positive immune balance regulation effect.This paper shows that the total extract, chloroform, ethyl acetate, N-butanol, and water fraction of The experiments follow the rules of the school’s animal ethics committee, which approves all experiments for the purpose of controlling and supervising animal experiments (protocol number 2015-0820).The following items were purchased from Beijing Solarbio Science and Technology Co., Ltd.: 3--2,5-diphenyltetrazolium bromide , dimethyl sulfoxide , phosphate-buffered saline , neutral red (Lot No. 925B047), and 2-[4-(2-hydroxyethyl)-1-piperazinyl]-ethanesulfonic acid . Fetal bovine serum was purchased from Gibco, lipopolysaccharide (LPS) was purchased from Sigma, and Astragalus polysaccharide injection was purchased from Henan Fuxing Animal Pharmaceutical Co., Ltd. (Lot. No.20140615). Cyclophosphamide  was purchased from Jiangsu Shengdi Pharmaceutical Co., Ltd. (Lot. No.16070425).Healthy Kunming (KM) mice (18–22g), were purchased from Hunan Jingleike Jingda Experimental Animal Co., Ltd., license number: SCXX (Xiang) K2016-0002.All the experimental samples were provided and prepared by Yunnan Provincial Key Laboratory of Entomological Biopharmaceutical R&D, Dali University.–1 with 1640 medium containing 10% fetal bovine serum. Next, 100 μL per well was inoculated on a 96-well plate and cultured at 5% CO2 and 37 °C. After 3 h, the unadherent cells were washed away with PBS to obtain purified peritoneal macrophages,After KM mice were sacrificed with cervical vertebra dislocation, macrophages were collected from the abdominal cavity, and the cell density was adjusted to 2 × 10–1 was seeded in 96-well plates and set cell control group (cells and culture medium only) and LPS model group . Respectively, sample groups  with LPS added  were placed in three wells each. After culturing at 37 °C in 5% CO2 incubator for 24 h, washed the plate with PBS and added 200 μL neutral red solution to each hole, incubated for 3 h. The supernatant was discarded and the cells were washed with PBS. Then 200 μL cell lysate  was added and the solution was left at 4 °C overnight. Finally, the absorbance was measured at 520 nm. The phagocytic index was calculated as follows  to obtain a red blood cell concentration of 2 million cells per microliter.Under aseptic conditions, blood was taken from the external jugular vein of healthy sheep, placed in an Erlenmeyer flask containing glass beads, and shaken for 30 min to remove fibrin. Next, PBS was added and the flask was stored in a refrigerator at 4 °C for later use. Before use, the sample was washed with normal saline and centrifuged for 5 min at 2000 r·minFresh blood was obtained from guinea pigs under aseptic conditions and the serum was separated and stored at –80 °C, then diluted with normal saline at 1:10 (v/v) before use.–1, while the experimental groups were given intragastric administrations of 400, 200, and 100 mg·kg–1. The medicine was administered once a day for 10 days. The model group was intraperitoneally injected 80 mg·kg–1 of CTX on the 7th day. Each sample group was immunized by an intraperitoneal injection of 0.2 mL 20% sheep red blood cell suspension on the 3rd, 5th, and 7th days of administrationThe immune function assay for normal mice was performed in the same manner as previously described. The mice were randomly divided into six groups: control group, model group, Astragalus polysaccharides (APS) group, and three experimental groups . There were six mice in each group, half male and half female. Each subject in the blank group was given the same quantity of normal saline. The APS group was intraperitoneally injected with APS 160 mg·kg–1 CTX and injected daily for three days to establish the model of immunosuppression in miceThe immune function assay for immunocompromised mice was performed as previously described. The grouping and administration were essentially the same as for the normal mice model. Except for the control group, from the 7th day of administration, each group was injected intraperitoneally with 0.1 mL of 80 mg·kgOne hour after the last administration, blood was obtained from the eyeballs of the mice, and 20 μL of whole blood was placed in the prediluted solution. The peripheral blood cells were determined by an animal blood cell analyzer in prediluted mode.–1, taken the serum and inactivated at 56 °C for 30 min. Diluted and placed in an EP tube, added 0.5 mL of 5% sheep red blood cells and 1.0 mL of diluted guinea pig serum, normal saline as the blank control. All the test tubes were kept at 37 °C for 10 min with water bath and then put into ice bath to terminate the reaction. Centrifuged and took 1.0 mL of the supernatant, added 3.0 mL of Drabkin’s reagent; took another test tube and added 0.25 mL of sheep red blood cell suspension and 3.75 mL of Drabkin’s reagent50)  21:The mice were sacrificed after blood collection, then the spleen and thymus were removed and accurately weighed to calculate the organ index of the immune organs .A one-way analysis of variance was performed with SPSS 19.0 software. After the homogeneity of the variance test, the experimental data with uniform variance were statistically analyzed by the pairwise comparison Fisher’s least significant difference method. Besides, the data with uneven variance was analyzed by the rank sum test. Values of p < 0.05 were considered statistically significant.B. rynchopetera were tested.Mouse peritoneal macrophages were used as the experimental object and LPS acted as a positive control. The phagocytic ability of macrophages was investigated by single sample stimulation and sample combined with LPS stimulationThe results are displayed in When stimulated with sample combined with LPS , while the positive drug APS group was significantly increased (p < 0.01). The EAF exhibited an increase, with the high and medium dose groups presenting a slight rising trend, but only the low-dose group displayed statistical significance (p < 0.05).50 of all experimental groups were significantly increased (p < 0.01), and the three-dose EAF groups were notably different from APS group (p < 0.01), reaching similar levels to the control group.In Blaps rynchopetera extract. Macrophages are multifunctional immune cells that play a key role in all aspects of the body’s immune response,in vitroFor the first time, the present study investigated the immunomodulatory effects of ,It has been previously shown that polysaccharides stimulate macrophage proliferation with low cytotoxicityIn vivo, the effects of EAF on the immune function of normal and immunocompromised mice were further investigated in terms of both cellular and humoral immunity. Astragalus polysaccharide is an extract of Astragalus membranaceus, which is rich in nutrients and has pharmacological effects such as enhancing immunity and improving antioxidant capacity. It has been widely used in clinical medicineThe results of this study show that APS has a significant effect on both normal and immunocompromised mice. It is noteworthy that EAF could significantly increase the immune organ indexes, the level of peripheral blood immune cells, and the half hemolysis value for immunocompromised mice. Conversely, it has a much smaller influence on normal mice. These results indicate that EAF has a remarkably protective effect on immunocompromised mice induced by CTX, and the regulatory mechanism may be different from that of APS.The test results of the normal group for both batches of experiments are also quite different, especially for the immune organ index. This reminds the necessity for using animals from the same batch in related subject research in future studies.In this study, experiments using neutral red assay revealed that all fractions can individually enhance the phagocytic ability of mouse peritoneal macrophages. The chloroform and EAF had a tendency to enhance the phagocytic ability of mouse peritoneal macrophages under combined stimulation with LPS. While EAF does not have a significant effect on the immune function of normal mice, it can significantly improve the state of low immunity caused by CTX, increase the level of immune cells, and enhance humoral immune function. Ethyl acetate fraction can regulate the balance of the abnormal immune state by stimulating immune organs and immune cells to produce immune response.


The genetic architecture of cancer has been delineated through advances in high‐throughput next‐generation sequencing, where the sequential acquisition of recurrent driver mutations initially targeted towards normal cells ultimately leads to malignant transformation. Myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) are hematologic malignancies frequently initiated by mutations in the normal hematopoietic stem cell compartment leading to the establishment of leukemic stem cells. Although the genetic characterization of MDS and AML has led to identification of new therapeutic targets and development of new promising therapeutic strategies, disease progression, relapse, and treatment‐related mortality remain a major challenge in MDS and AML. The selective persistence of rare leukemic stem cells following therapy‐induced remission implies unique resistance mechanisms of leukemic stem cells towards conventional therapeutic strategies and that leukemic stem cells represent the cellular origin of relapse. Therefore, targeted surveillance of leukemic stem cells following therapy should, in the future, allow better prediction of relapse and disease progression, but is currently challenged by our restricted ability to distinguish leukemic stem cells from other leukemic cells and residual normal cells. To advance current and new clinical strategies for the treatment of MDS and AML, there is a need to improve our understanding and characterization of MDS and AML stem cells at the cellular, molecular, and genetic levels. Such work has already led to the identification of promising new candidate leukemic stem cell molecular targets that can now be exploited in preclinical and clinical therapeutic strategies, towards more efficient and specific elimination of leukemic stem cells.  The recognition that cellular, genetic, and molecular features of cancer allow for segregation of cancer subgroups with different diagnosis, disease progression, and response to treatment, has facilitated development of therapeutic strategies tailored towards patients most likely to respond while reducing the risk of life‐threatening side effects , 2. AdvaIn light of the identity of cancer stem cells  being defined by their functional characteristics, significant efforts have been directed towards the development of experimental assays for assessment of self‐renewal and tumor‐propagation potentials from human patient material . CombineMyelodysplastic syndromes (MDS) and AML are two related myeloid malignancies, which each encompass a heterogeneous group of patients with regard to clinical features, genetics, prognosis, and treatment , 12. TheDNMT3A, the most commonly mutated gene in CH individuals, is also recurrently mutated in AML patients where DNMT3A is frequently predicted to be the first/initiating event . Th. Th11]. As incorporated into the diagnostic criteria, cytopenia impacting one or more of the erythroid, myeloid, or platelet lineages is a common feature in MDS, with anemia being the most prevalent, present in 80% of patients , 62. PanDNMT3A, TET2, and ASXL1 are frequently mutated along with mutations in splice factor genes [TP53 mutations [Although conventional chemotherapy has been used to treat high‐risk MDS , 74, itsor genes , 15, 16.or genes , 77, 78.or genes . Throughutations , 81, 82.FLT3 mutated AML) and IDH1/IDH2 inhibitors (IDH1/IDH2 mutant AML) are under development or in clinical use [Clone‐reductive therapies are critical strategies that can stabilize the disease, delay disease progression, and reduce the disease burden over a longer period, thereby prolonging the quality of life and lifespan. Such effects can be achieved by therapy that targets both bulk and leukemic stem cells, as well as by therapy that targets the bulk leukemia preferentially, where the latter would lead to a likely relapse. In addition to hypomethylating agents, treatments targeting genetic features of the malignant clone, such as FLT3 inhibitors  of an antibody, combination therapies may be synergistic or detrimental; for example hypomethylating drugs have been shown to enhance the ADCC activity of NK cells, of importance if an antibody uses effector cells to elicit killing of their target cells , whereaswww.clinicaltrials.gov). Many of these trials will read out in the near future, hopefully, leading to better treatment outcomes for patients diagnosed with MDS and AML and to increased insights into the biology of leukemia stem cells and the feasibility of using antibody based therapies directed at targets expressed on their cell surface.Of the cell surface markers discussed in previous paragraphs, only an antibody against CD33 (gemtuzumab) has received FDA approval . A CD123Given the success of chimeric antigen receptor (CAR) T‐cell therapy mainly in B‐cell malignancies, there is an increasing interest to use this technology to also target MDS and AML stem cells. The fact that B‐cell malignancies are amenable to this approach is not surprising as several cell surface markers  are highly specific for the B‐cell lineage and, with proper precautions and supportive treatment, an individual can live without normal B cells. In contrast, this is not the case for myeloid lineage as such cells are required to sustain the life of an individual. Many innovative approaches using the CAR‐T‐cell technology are currently being tested preclinically and in clinical trials . Given tFLT3 tyrosine kinase domain has been productive for the treatment of FLT3‐mutated AML [FLT3‐mutated AML cells are particularly sensitive towards targeted disruption [BCR::ABL1 fusion product in CML [Some of the recurrent somatic oncogenic mutations that drive MDS and AML provide unique opportunities for targeting the malignant cells with high specificity Fig. . One claated AML . In lighsruption , 117. Tht in CML , 120. Ast in CML .IDH1 and IDH2 lead to reduced production of α‐ketoglutarate (α‐KG), the key product catalyzed by the normal IDH1/IDH2 enzymes, and production of a novel oncometabolite, 2‐hydroxygluterate (2‐HG) [Somatic mutations in e (2‐HG) , 123. See (2‐HG) , 125. The (2‐HG) .casein kinase 1A1 (CSNK1A1) [Csnk1a1 demonstrated that although haplo‐insufficiency for CSNK1A1 promotes HSC expansion, in line with the competitive advantage of del(5q) MDS stem cells, homozygous deletion of Csnk1a1 results in HSC failure [CSNK1A1. However, lenalidomide is not sufficient to eliminate all del(5q) MDS stem cells, as a significant proportion remain even in the bone marrow of patients in complete clinical and cytogenetic remission [Heterozygous deletion of an essential gene can provide a therapeutic window to kill malignant cells, as is the case in del(5q) MDS patients treated with lenalidomide , 96. DelCSNK1A1) . Mouse s failure . Interes failure . These femission .Somatically acquired genetic lesions can alter cell state in a manner that makes leukemia cells susceptible to a pharmacologic intervention relative to normal cells, a concept termed synthetic lethality. This may be the basis of the activity of hypomethylating agents and venetoclax in myeloid malignancies , 131. MuSomatically acquired mutations can also open future possibilities for immunological targeting of AML and MDS cells , 137. MuBCR::ABL1 fusion with tyrosine kinase inhibitors in CML and del(5q) mutated cells with lenalidomide, the leukemic stem cells are particularly challenging to eliminate fully. Effective elimination of the leukemic stem cells will likely require combination therapies that carefully consider the duration, dose, and timing of treatment.A major challenge is that MDS and AML are genetically heterogeneous, and patients have multiple sequentially acquired mutations. Targeting a mutation acquired early in disease development would potentially eliminate all mutant cells, while targeting a mutation acquired late in disease ontogeny might only have an effect on a subclone. As revealed by targeting of the Genomic profiling of MDS and AML has become clinical routine in many countries and to a large degree influences the clinical management of MDS and AML patients. This has led to the development of new promising therapies exploiting the altered genetic and cellular composition of MDS and AML, yet most patients today lack a treatment that is specifically designed for the underlying molecular cause of the disease. In addition, disease progression, relapse, and treatment‐related mortality still remain a challenge for the treatment of MDS and AML patients. Leukemic stem cells, required for the propagation of the leukemia, are often particularly challenging to eliminate and have evolved mechanisms to escape therapeutic targeting. New therapeutic strategies are, however, being tested in preclinical and clinical studies, including small molecules and antibody‐based therapies, hopefully improving our ability to specifically target the leukemic stem cells. The impact of treatment can be observed by tracking the leukemic stem cells in the patients following treatment, but in several cases this is limited by our ability to distinguish leukemic stem cells from other normal or leukemic cells and lack of sensitivity of available methods. Advances in single cell technology, combining genetic and molecular profiling of single cells can in the future facilitate better understanding of cells evading therapy, as well as allow clinical surveillance and detection of rare, therapy‐resistant leukemic stem cells prior to relapse.B. L. E. has received research funding from Celgene, Deerfield, Novartis, and Calico and consulting fees from GRAIL. B. L. E. is a member of the scientific advisory board and shareholder for Neomorph Therapeutics, TenSixteen Bio, Skyhawk Therapeutics, and Exo Therapeutics. T. F. is a cofounder and board member and owns shares in Cantargia AB. All the other authors have no conflict of interest to declare.Conceptualization: all authors; Writing – original draft: Petter S. Woll and Tetsuichi Yoshizato;Writing – review and editing: all authors.


Soil waterlogging is among the major factors limiting the grain yield of winter wheat crops in many parts of the world, including the middle and lower reaches of the Yangtze River China. In a field study, we investigated the relationship between leaf physiology and grain development under a varying duration of post-flowering waterlogging. A winter wheat cultivar Ningmai 13 was exposed to soil waterlogging for 0 (W0), 3 (W3), 6 (W6), and 9 d (W9) at anthesis. Increasing waterlogging duration significantly reduced flag leaf SPAD  values and net photosynthetic rate (Pn). There was a linear reduction in flag leaf Pn and SPAD as plant growth progressed under all treatments; however, the speed of damage was greater in the waterlogged leaves. For example, compared with their respective control (W0), flag leaves of W9 treatment have experienced 46% more reduction in Pn at 21 d after anthesis (DAA) than at 7 DAA. Increasing waterlogging duration also induced oxidative damage in flag leaves, measured as malondialdehyde (MDA) contents. The capacity to overcome this oxidative damage was limited by the poor performance of antioxidant enzymes in wheat leaves. Inhibited leaf Pn and capacity to sustain assimilate synthesis under waterlogged environments reduced grain development. Compared with W0, W6 and W9 plants experienced a 20 and 22% reduction in thousand grain weight (TGW) in response to W6 and W9, respectively at 7 DAA and 11 and 19%, respectively at 28 DAA. Sustained waterlogging also significantly reduced grain number per spike and final grain yield. Averaged across two years of study, W9 plants produced 28% lesser final grain yield than W0 plants. Our study suggested that wheat crops are highly sensitive to soil waterlogging during reproductive and grain filling phases due to their poor capacity to recover from oxidative injury to photosynthesis. Management strategies such as planting time, fertilization and genotype selection should be considered for the areas experiencing frequent waterlogging problems. Agricultural production is currently facing remarkable challenges in feeding a rapidly growing world population under diminishing natural resources . This isThe dominant monsoon climate in this region, especially from March to May, often carries abundant precipitation with an erratic spatial and temporal distribution . ConsequThe negative effects of waterlogging on plants are initiated by soil oxygen deficiencies . Under oComparing the performance of wheat genotypes in response to soil waterlogging (21 d) during different developmental phases, i.e., jointing, flowering, and grain filling, Sustained carbohydrate and nutrient supplies are essential for wheat grains during the critical phases of grain development . Soil waField experiments were conducted at the experimental station of Anhui Agricultural University  in Lujiang County, Hefei City, Anhui Province, during 2015–16 and 2016–17. The region has a subtropical humid monsoon climate with an average annual precipitation, sunshine duration, and frost-free period of 1188.1 mm, 2209.6 h, and 238 d, respectively. The monthly precipitation during growing seasons is shown in Each experimental plot (2.4 m × 5.0 m) consisted of 13 rows with 20 cm spacing to achieve a target population of 300 plants per square meter. The treatments were replicated thrice in randomized complete block designs. The plot edges were insulated from each other with a plastic frame made of polyvinyl chloride. The plastic frame was buried 40 cm deep and extended 20 cm on the ground. The treatment plots were artificially waterlogged when 50% of the plants in the field passed flowering . TA major winter wheat cultivar in the middle and lower reaches of the Yangtze River China ‘Ningmai 13’ was used as a test cultivar. The seeds were sown on 8 November 2015 and 11 November 2016.−1, 75 kg P2O5 ha−1, and 150 kg K2O ha−1. All P and K fertilizers and 70% of N fertilizers were applied before sowing, and the remaining N fertilizer was top-dressed at jointing. Wheat plants were harvested on 24 May 2016 and 27 May 2017.All plots were supplied with 225 kg N haLeaf physiological data were recorded from all the plots at 7, 14, and 21 DAA. For this purpose, five tillers per replicate were tagged at flowering, and the same flag leaves were used for measuring SPAD and Pn.The SPAD value was measured non-destructively using SPAD 502 Meter . For each measurement, five SPAD readings were obtained and averaged.2 concentration of about 385 mol, and the reading of the light source was 1,200 mol·m−2·s−1. All measurements were made between 9:00 and 11:00 on days with full sunlight  at a COsunlight .For the destructive measurement, 15 tillers per replicate were tagged at flowering. Flag leaf samples were collected from the tagged tiller (5 tillers each time) at 7, 14, and 21 d after flowering. The sampled leaves were pooled and stored in liquid nitrogen until analysis. Catalase (CAT) and superoxide dismutase (SOD) activities and MDA content were determined according to For grain yield measurements, emerging flowering spikes were tagged on the same day. Twenty tagged spikes from each experimental plot were sampled at 7-day intervals from the beginning of anthesis to maturity. The grain filling rate was estimated from the accumulation of dry grain weight. At each sampling date, grains were separated from the glumes and dried at 70°C until a constant weight was reached. The total number of grains was determined, and their dry weight was recorded.The number of spikes and grains per spike were investigated at the mature stage, and each plot was separately threshed, weighed, and converted into the yield when the water content was 13%.α = 0.05 to identify significant differences among treatments. Multiple comparisons were made using the least significant difference test with α = 0.05 to determine significant differences among treatments. The Origin9.1  was used to show differences in SPAD values, Pn values, MDA content, SOD and CAT activities and TGW.Data were subjected to ANOVA . The ANOVA used a level of significance of Increasing waterlogging duration from 3 to 9 d significantly reduced leaf SPAD values at 14 and 21 DAA, although there was no significant impact of waterlogging duration on these traits at 7 DAA . FurtherShort-term waterlogging (W3) had no significant effect on the net photosynthetic rate (Pn) of flag leaves at 7 DAA, but it significantly reduced leaf Pn at 14 and 21 DAA . W6 and Increasing waterlogging duration significantly reduced SOD activity in flag leaves of wheat during all the tested developmental stages . Also, SDuring the two tested growing seasons, increasing waterlogging duration significantly reduced CAT activity in flag leaves during all developmental phases . CAT actDamage to flag leaf lipid membrane calculated in terms of MDA contents increased as the plant growth progressed with each subsequent measurement . IncreasDuring all the tested developmental stages, W3 had no significant effect on TGW, but W6 and W9 significantly reduced TGW during all developmental phases. This waterlogging-induced reduction in TGW was intensified as plant growth progressed. W6 and W9 had no significant effect during earlier and middle stages of grain development ; however, the TGW of W9 was significantly lower than that of W6 at 28 DAA . CompareTo find the relationships between the analyzed parameters, a principal component analysis (PCA) was performed. PCA analysis allows for the determination of variables that exert the greatest impact on the appearance of individual components. The presented PCA facilitates the interpretation of the impact of Pn value, MDA content, and antioxidant enzyme activities on TGW. Two main components derived from the calculations were selected; they allowed for the explanation of 93.8% of the total variance of analyzed variables. The first main component contains 81.6% of the information about the tested samples represented by variables, while the second main component contains 12.2% of the information .Coefficients of PC1 (eigenvectors) leaf Pn, SPAD, and antioxidant enzymes were positive, while MDA was negative. Significantly higher values of eigenvectors for SPAD and MDA indicate superior leaf SPAD and lower MDA contents are a good indicator of plant performance under waterlogging. This separated the plants on the basis of waterlogging treatment in the same direction. W0 plants were grouped together (the top right hand side of the axis), indicating a strong association of TGW with Pn and other leaf physiological traits. With the increasing waterlogging duration, the plants were grouped to the left hand side of the axis, indicating higher MDA level could reduce grain size. The eigenvectors values of PCA indicated that PC2 was an index of the subtle differences in Pn and MDA contents, and thus could explain the variable grouping of plants based on the waterlogging duration.Waterlogging treatments had no significant effect on the total number of spikes in this study, but other grain yield components such as kernel numbers, TGW, and total grain yield all were significantly affected by W6 and W9 treatment . W3 had In this study, increasing waterlogging duration significantly reduced leaf greenness , and the damage accelerated as the plant progressed. Flag leaf SPAD value is considered a good indicator of plant health . EarlierLeaf chlorophyll is a site for photosynthesis and assimilates production. In plants, the assimilation process is strongly associated with leaf age, leaf chlorophyll content, and the activity of enzymes . DevelopIn this study, increasing waterlogging duration significantly damaged the lipid membrane of flag leaf tissues in wheat. Excessive ROS and oxidative damage are commonly observed phenomena in stressed plants. These ROS can harm cellular membrane permeability and thus inhibit plant growth, development, and final yield . Under wUnregulated cellular ROS can increase the peroxide level , damaginIn this study, waterlogging significantly reduced grain yield components, including TGW and grain number per spike. This yield loss was strongly associated with waterlogging duration, i.e., grain yield loss increased from 3.7 to 28.8% by increasing waterlogging duration from 3 to 9 d. Significant grain yield loss, i.e., up to 40%, has been recorded in wheat crops in response to sustained waterlogging . Soil waIn this study, grain yield loss increased from 3.7 to 28.8% by increasing waterlogging duration from 3 to 9 d. This grain yield loss was due to oxidative stress and lipid membrane damage in flag leaves, which accelerated leaf senescence and inhibited photosynthesis, reducing assimilates supplies to the developing wheat grains. This study found that short-term waterlogging (W3) reduced current sources (photosynthesis) during later phases of grain development only (14 and 21 d after anthesis), resulting in no significant reduction in TGW. However, the impact of W6 and W9 significant on Pn during the first week of waterlogging and thus these treatments reduced TGW. Our study suggested that sustain carbohydrates supplies are critical for developing wheat grains during early phases development. Crop management techniques should be adjusted to sustain assimilates supplies during this critical period, particularly under stressed environments.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.SM and ZH contributed to conception and design of the study. JH organized the database. SM, JH, and YW performed the statistical analysis. SM wrote the first draft of the manuscript. All authors contributed to the article and approved the submitted version.This research was supported by the National Natural Science Foundation of China (31801287), the grants from the National Key Research and Development Program of China (2016YFD0300405 and 2017YFD0301305), and the Project of China Scholarship Council (201908775002).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.


ACC presented a significantly higher Ang1 and Ang2 expression when compared to ACA. Tie1 expression was higher in ACC with venous invasion and in patients with shorter overall survival. In conclusion, although none of these biomarkers showed to be useful for ACT diagnosis, the Ang–Tie pathway is active in ACT and may play a role in regulating ACT angiogenesis.The differential diagnosis between adrenocortical adenomas (ACAs) and adrenocortical carcinomas (ACCs) relies on unspecific clinical, imaging and histological features, and, so far, no single molecular biomarker has proved to improve diagnostic accuracy. Similarly, prognostic factors have an insufficient capacity to predict the heterogeneity of ACC clinical outcomes, which consequently lead to inadequate treatment strategies. Angiogenesis is a biological process regulated by multiple signaling pathways, including VEGF and the Ang–Tie pathway. Many studies have stressed the importance of angiogenesis in cancer development and metastasis. In the present study, we evaluated the expression of VEGF and Ang–Tie pathway mediators in adrenocortical tumors (ACTs), with the ultimate goal of assessing whether these molecules could be useful biomarkers to improve diagnostic accuracy and/or prognosis prediction in ACC. The expression of the proteins involved in angiogenesis, namely CD34, VEGF, VEGF-R2, Ang1, Ang2, Tie1 and Tie2, was assessed by immunohistochemistry in ACC ( Adrenocortical tumors (ACTs) originate in the adrenal cortex and affect 3 to 10% of the human population . In contAngiogenesis represents the growth and development of new blood vessels from pre-existing vasculature, an important phenomenon in tumor biology which is involved in tumor progression . SimilarVEGF is the most potent inductor of angiogenesis. VEGF activates the vascular endothelial receptor 2 (VEGF-R2) that, in turn, induces angiogenesis by promoting proliferation, migration and cell survival. The Ang–Tie signaling cascade includes angiopoietin 1 (Ang1) and 2 (Ang2) and the transmembrane receptors Tie1 and Tie2. This pathway seems to act as VEGF co-adjuvant by controlling later stages of angiogenesis, while regulating vascular permeability and remodeling. The VEGF status of adrenocortical tumors was previously described in Reference . HoweverIn the present work, we aimed to evaluate the expression of molecules that participate in VEGF and Ang–Tie pathways in ACT and its potential use for fine-tuning differential diagnosis and/or prediction of ACC prognosis.CD34 immunochemistry (IHC) staining was used to evaluate ACT tumor blood vessel density a–c.p < 0.01) and non-functioning adrenocortical adenoma (ACAn) . No differences were observed between ACAn and ACAc (The percentage of CD34 staining area was significantly lower in ACC (4.023 ± 0.408%) when compared to both adrenocortical adenoma with Cushing syndrome (ACAc)  (All ACTs expressed VEGF with a variable expression pattern within the tumor cells. VEGF expression was found in the cytoplasm and nucleus in 38% of ACC and ACAn . VEGF ex = 0.09) .p < 0.05)  b. No difAng1 was found to be expressed in all ACTs a–c. ACC p < 0.05)  d.Tie1 expression was present in all ACTs, except in a single ACAn g–i. No sp < 0.05)  f. In addNo significant correlations were found between the angiogenic markers in ACC, ACA and tumor or patient characteristics .p = 0.021)  Stage; and presence or absence of distant metastasis, capsule, venous and sinusoids invasion . A highe= 0.021) . No addi= 0.021) .CD34 was demonstrated to have good accuracy for the differential diagnosis between ACC and ACA on ROC curve analysis. This molecular marker was particularly accurate in differentiating ACC and functioning ACA (ACAc). In addition, the Tie2 receptor also proved to be a good marker in distinguishing ACC from ACAc b.Despite the differences observed in molecular expression when comparing ACA and ACC, the remaining biomarkers analyzed presented low accuracy in regard to distinguishing ACC from ACA .There was no significant difference in angiogenic markers expression nor VEGF expression pattern in ACC according to patient overall survival. A higher Tie1 expression was found in most patients with lower survival .The poor specificity of pathologic findings used to diagnose ACC and predict disease prognosis are major contributors to diagnosis inaccuracy and inappropriate treatment interventions. In the present study, the expression of VEGF and Ang–Tie pathways’ molecular intermediates of ACT was characterized, while the potential for increasing the diagnosis and/or prognosis prediction accuracy was evaluated.In our study, CD34 expression was used to assess vascular density. Although a high vascular density was globally present in ACT, this was significantly lower in ACC when compared to ACA.Previous studies on ACC and ACA vascular density yielded inconsistent results ,12,13,14In addition to vascular density analysis, the status of VEGF and Ang–Tie pathways in ACT was assessed, thus enabling us to demonstrate that both VEGF and Ang–Tie pathways are expressed in ACT.In ACC cells, VEGF expression can depict two different location patterns, in cytoplasm only or in both the nucleus and cytoplasm. Nuclear expression was found in 38% of ACC and of ACAn, whereas, in the remaining tumors, immunostaining was exclusively present in the cytoplasm. VEGF nuclear expression in other tumor types, such as breast cancer and colorectal cancer, has been previously reported ,16. HoweIn our study, ACAc presented a significantly greater VEGF-R2 expression when compared to ACAn. VEGF is highly expressed by the adrenal cortex and required to maintain a dense and fenestrated vasculature necessary for efficient steroid secretion . TherefoThis study is also the first to report that the Ang–Tie pathway signaling molecules are expressed in ACT. Ang2 can play different roles in tumor angiogenesis. In the presence of VEGF, Ang2 acts synergistically with VEGF to promote angiogenesis, while, in the absence of VEGF, Ang2 induces blood vessel regression . Since VACC with venous invasion also presented a greater Tie1 expression when compared to ACC without venous invasion. This finding might be potentially explained by endothelial wall disruption, which results in loss of the venous endothelium quiescent state, which, in turn, triggers Tie1 expression . In addiDespite the differences observed, neither angiopoietins nor tie receptors demonstrated to be useful biomarkers for the differential diagnosis of ACT on ROC curve analyses.n = 43), including ACC (n = 22), ACAc (n = 8) and ACAn (n = 13). A summary of patients’ and tumors’ characteristic is given in Adrenal tissue was obtained during adrenalectomy from patients with ACTs . Tissue sections were deparaffinized in xylene and hydrated in downgraded alcohols before undergoing antigen retrieval, as described in 2O2)  in methanol to inhibit endogenous peroxidase for 15 min. For Ang2, tissue sections were also incubated with 10% bovine serum albumin (BSA) at room temperature for 30 min to block non-specific reactions. Then tissue sections were incubated overnight at 4 °C with the primary antibody. The detection of the immune reaction was performed by incubation for 60 min with the commercial Dako Real™ EnVision™ Detection System . Then 3,3′-diaminobenzidine (DAB) was used as chromogen, and hematoxylin as nuclear counterstaining.IHC was performed in 3 µm formalin-fixed paraffin embedded tissue sections mounted on adhesive microscope slides . The tumor area was delimited by a pathologist, and adjacent tissue and capsule were not included in the analysis. IHC images were analyzed by using the software ImageJ  that allows for the detachment of the stained area from each histological image based on the Red–Green–Blue (RGB) system. The percentage of the stained area for each protein was calculated through the ratio between the stained and the total tissue area. Unspecific staining that included areas of necrosis and fibrosis was excluded. VEGF expression presented two different patterns, with cell staining occurring both in the cytoplasm and nucleus, or in cytoplasm only. Thus, VEGF expression pattern evaluation was first identified by direct observation and then quantified in tumors that presented cytoplasm and nuclear expression and cytoplasm expression only.Slides were scanned for each marker, using the image-acquisition Olympus2. Correlations between continuous variables were evaluated by using the Pearson test or the Spearman test, depending on the variables. The Kaplan–Meier method was used to compare the overall survival of patients depending on the VEGF expression pattern. For the remaining markers, the Cox regression model, adjusted for age, was used for overall survival analyses.All ordinal data are represented as mean ± standard error of the mean (SEM). To evaluate the variables’ normality, the D’Agostinho–Pearson test was used. For variables that passed this test, the one-way ANOVA test with the post hoc Tukey was used to compare the means of 3 groups, and the t-test was used to compare 2 groups. For variables that did not pass the normality test, the Kruskal–Wallis with a post hoc Dunn’s was used to compare 3 groups, and the Mann–Whitney test was used to compare 2 groups. The two different VEGF expression patterns were compared by χp < 0.05 was considered statistically significant.The receiver operating characteristic (ROC) area under the curve (AUC) was used to determine the angiogenic markers’ diagnostic accuracy. All statistical analyses were performed by using the GraphPad Prism (version 8.01), except for the Cox regression analysis, which was performed by using the SPSS software (version 26.00 for Windows). A This is the first report to describe the expression of Ang–Tie pathway in ACT. When compared to benign tumors, ACC presented a lower vascular density but a higher Ang2 expression, a biomarker related to vascular permeability and cell spreading. Additionally, Tie1 expression was higher in ACC with venous invasion and pertaining to patients with lower overall survival. Although functional and mechanistic studies are still needed to validate these results, our data support a role for the Ang–Tie pathway in ACT angiogenesis.


Osteoarthritis is a source of chronic pain and disability. Dietary supplements have been shown to be a more secure option than NSAIDS. Particularly, the eggshell membrane has demonstrated efficacy in relieving joint pain and stiffness. A clinical trial was designed in which three groups were assigned to two different doses of this supplement and compared to a placebo control group. The primary outcome variable was knee pain, which was assessed using a visual analogue scale. Secondary outcome variables were knee functional ability, quadriceps muscle strength , and quality of sleep. All groups showed a significant decrease in pain perception, although maximum values were obtained in the high-dose group. Isokinetic and isometric trials showed a significant increase in strength in the high-dose group. Eggshell membrane showed the potential to reduce pain and stiffness symptomatology. Here, for the first time, two quantitative variables (mobility and strength of knee joint) were used to accurately evaluate changes in the quality of life of subjects affected by knee joint pain. The results of this study indicate a dose-dependent response, which should be taken into account for later use in therapeutics to establish the correct dosage. Increasing life expectancy has led to structural changes in current human populations . Ageing OA is the most prevalent joint disorder and source of chronic pain and disability in developed countries ,6. This Acute pain is a commonly associated symptom; therefore, pharmacotherapy is indicated as treatment through the use of analgesics and non-steroidal, anti-inflammatory drugs (NSAIDS) ,9. HowevAmong them, eggshell membrane has demonstrated efficacy in relieving joint pain and stiffness ,17,18,19Therefore, the objective of this study was to determine the efficacy of a food supplement extracted from the internal membrane of the eggshell on joint functionality  and perceived pain  in individuals diagnosed with OA, after a consumption period of eight weeks. Two doses were tested in order to evaluate efficacy in a placebo-controlled clinical trial.A randomised, controlled, double-blind, single-centre clinical trial was designed, in which three groups were assigned to different doses of an internal membrane, eggshell-based supplement . This stThe trial design followed CONSORT guidelines. Written, informed consent was signed by all participants after approval by the University’s Institutional Science Ethics Committee. Inclusion criteria were as follows: subjects diagnosed with arthrosis with functional grades I–III according to criteria from the American College of Rheumatology, Atlanta; persistent knee pain associated with this pathology, with initial punctuation of at least 30 mm in a visual analogue scale of 10 cm; and absence of chronic treatment with narcotics, NSAIDs, or immunosuppressants. Exclusion criteria were as follows: terminal disease; known chronic inflammatory diseases affecting the musculoskeletal system ; other serious illnesses that would limit the execution of aerobic or resistance exercises ; body mass index (BMI) above 32; subjects who, at the time of the study, were being treated with glucosamine, chondroitin sulphate, collagen, or hyaluronic acid infiltrations, or consuming any supplement indicated for joint health; subjects under pharmacotherapy ; known allergy to eggs; pregnant or breastfeeding women; and incapacity to understand informed consent.® eggshell membrane  in vegetarian capsules that were stored in closed containers at ambient temperature. The control group consumed a placebo based on encapsulated maltodextrin matching the weight and aspect of treatment. Eggshell membrane extract was composed mainly of proteins (>90%), fibrous collagen types I, V, and X (<13%), and elastin (<4–5%). The extract also contained glycosaminoglycans, such as chondroitin sulphate (<2%); dermatan and keratin sulphates (<1%); and hexosamines, such as glucosamine (<2%), hyaluronic acid (<2%), IGF-1 (12 ng/g), TGF-beta (0.75–7.23 ng/g), calcitonin (10–25 ng/g), and progesterone (0.3–0.33 ng/g). Clinic visits were scheduled for subjects at study initiation and at the end of 8 weeks following the onset of treatment. Compliance with the treatment was evaluated during the last visit via patient interview and by counting the number of unused capsules still remaining. Lifestyle variables included a dietary recall interview of the previous 3 days before starting the nutritional supplement and the last 72 h before the end of the 8-week consumption of the product. The level of physical activity was recorded using the Global Physical Activity Questionnaire (GPAQ), and results were expressed as MET-min/week. Specific rules were given to subjects about maintaining the same level of physical exercise during the trial. Weight was registered via bioimpedance.Treatment consisted of once-daily oral ingestion of 300 mg (low dose) and 500 mg (high dose) of ESMThe primary outcome variable was knee pain which was assessed using a visual analogue scale (VAS) of 10 cm, defining extreme limits such as ‘no pain at all’ and ‘pain as bad as it could be’ . Values Secondary outcome variables were knee functional ability, quadriceps muscle strength , and quality of sleep.Functional ability was evaluated using the Western Ontario and McMaster Universities Osteoarthritis (WOMAC) index, through an adapted Spanish version of the WOMAC questionnaire . This quMuscle strength was determined using a Biodex System 3 Dynamometer  in order to assess isometric (straining your muscles without moving or bending your joints) and isokinetic  strength of the knee flexors and extensors. It is important to note that changes in strength should be associated with changes in perceived pain and not with changes in the physical condition of the muscle. Trials involved two maximum isokinetic and two continuous maximum isometric repetitions at 60°/s and 90°/s on their right leg, respectively. Isokinetic trials collected information about muscle peak torque ; total work of the maximum repetition ; and total work . Isometric trials assessed muscle peak torque (PT/BW) and maximum average peak torque . Verbal encouragement was provided during the tests, and adequate rest and recovery times were provided between contractions in order to minimise fatigue.Quality of sleep was assessed by the Pittsburgh Sleep Quality Index (PSQI) , which hANOVA with repeated measures was used to compare variables that were obtained at the beginning and end of this trial. All variables were checked for normality. Homogeneity of variables among groups was also checked in the baseline in order to avoid confounding variables. Bonferroni correction was used for all comparisons between intervention groups and control. Type I error rate was set at α = 0.05. Analyses were performed by using SPSS statistical software v.21.0 .A total of 80 out of 120 subjects assessed for eligibility were randomised , registering 4.75 ± 1.16 points (control), 4.69 ± 1.18 points (low dose), and 5.23 ± 1.32 points (high dose). A progressive decrease in the evolution of pain can be observed for all groups, and the lowest values on the scale were achieved in the last week for all groups (p > 0.05). The other two groups registered a significant decrease in perceived pain, decreasing to 3.13 ± 1.43 points (mild to moderate pain) in the low-dose group, and decreasing to 2.98 ± 1.51 points (mild pain) in the high-dose group (p < 0.014).The evolution of pain perception as soon as a subject awoke during the study period can be also followed in l groups . The conse group . Comparip > 0.1). Despite the fact that all three groups showed a significant decrease in the WOMAC scale score (The WOMAC results were homogeneous among the three groups at the beginning of this trial . The conle score , both grle score .Isokinetic and isometric trials showed no significant differences at the beginning of this study for all variables included . AlthougPittsburgh test scores provided similar scores at the beginning of this trial for all three groups , and no Subjects did not report any adverse events related to the consumption of the product administered.Pain due to osteoarthritis is known to induce long-term consequences on health as a consequence of a lack of exercise . In factEggshell membrane showed the potential to reduce such symptomatology. Although all of the groups registered a decrease in perceived pain, both doses used in this study induced a bigger reduction than in the control group, indicating a relationship between eggshell membrane consumption and reduction in pain. Such results are compatible with previous studies that demonstrated the efficacy of this nutraceutical to alleviate knee pain, symptomatology, and joint functionality ,17,18,19Reduction in perceived pain was decremental and was observed during the entire study period, as registered by the information collected as soon as the subject awoke in the morning. Such a variable registered a significant weekly evolution of pain through the study for the high dose, showing a final reduction of 0.69 points more than the low dose . Those rIn fact, such an observation could be in agreement with the results obtained for muscle strength. All variables studied here registered an increase in both consumption groups, although a higher dose-dependent effect was registered than for other tests . IsokineAlthough previous studies addressed the study of the efficacy of eggshell membrane as a supplement to alleviate knee joint pain ,16,17,18


Furthermore, they improve the mechanical and thermal properties of the polymer and ceramic solid-state electrolytes (SSEs). Overall, the enhancements endowed by GBMs will address the challenges that are stunting the proliferation of SSBs. Solid-state batteries (SSBs) have emerged as a potential alternative to conventional Li-ion batteries (LIBs) since they are safer and offer higher energy density. Despite the hype, SSBs are yet to surpass their liquid counterparts in terms of electrochemical performance. This is mainly due to challenges at both the materials and cell integration levels. Various strategies have been devised to address the issue of SSBs. In this review, we have explored the role of graphene-based materials (GBM) in enhancing the electrochemical performance of SSBs. We have covered each individual component of an SSB  and highlighted the approaches using GBMs to achieve stable and better performance. The recent literature shows that GBMs impart stability to SSBs by improving Li SHE)  polymer was demonstrated . The ele0 cycles d. The ca0.5 for 1 vol% to 2.4 MPa.m0.5 for 5 vol% , ceramic SSEs do face problems such as low fracture toughness, especially when operated under stack pressures. Any tiny microscale fracture in the bulk of electrolyte provides paths for Li dendrites to sneak through that poses a risk of cell short circuit ,90. It ir 5 vol% a. It wasr 5 vol% c,d, wherr 5 vol% e that th7P3S11 SSE particles results in a stable and efficient all-solid-state battery performance. The synthesis of GO@LPS materials was performed using a simple facial technique where GO and LPS were mixed in acetonitrile followed by an annealing step as shown in + ions, GO is reduced to rGO; however, since a very small amount is added (~1 wt%), no percolation network is established. This was confirmed when the electronic conductivity of the electrolyte was 5 orders of magnitude lower than the ionic conductivity. Stable Li stripping/platting voltage profiles were observed for more than 600 h, unlike the uncoated LPS that short-circuited even before reaching 50 h of cycling. The Li|GO@LPS|LCO cells performed much better than cells based on only LPS electrolyte as shown in + ion flux at the interface.Another work explored a different role of GO in a sulfide LPS SSE . Due to −1. It is well known that sulfur as an electrode in liquid electrolytes is problematic due to the dissolution and “shuttle effect” of polysulfides, which greatly decrease cycle life and specific capacity. Replacing the liquid electrolyte with the SSEs can somehow address the polysulfide dissolution, and improve the safety and cycle stability. However, integrating sulfur cathodes with SSEs can increase the cell resistance due to poor electrode|electrolyte contact, although, some solutions have been found for this problem [GBM-enhanced cathode materials have performed well when integrated with different SSEs. Among various cathodes, sulfur is a popular choice due to its high theoretical capacity of 1672 mAh g problem . In addi problem . The ion problem ,99. Gene2S-24%P2S5-1%P2O5 electrolyte layer was inserted between the LGPS electrolyte layer and the lithium anode to avoid any undesired reactions between lithium metal and LGPS (−1 at 1.0 C)  b and lon2 gel polymer electrolyte (GPE) film. The cell exhibited a high initial specific discharge capacity (809 mAh g−1) and maintained a reversible discharge capacity of 413 mAh g−1 after 50 cycles under a 0.2 C rate. The cycling performance of the cell indicates that the combination of the S/GNS composite cathode and GPE plays a significant role of retarding diffusion of the polysulfides out of the cathode and suppressing their transport towards the anode side. The rate capability performance was also investigated, and the results indicated that the cell delivers a reversible discharge capacity of 638 mAh g−1 at 0.1 C. The high-rate operation was ascribed to the enhanced conductivity of the GNSs, which act as nano-current collectors, and the good ionic conductivity of the GPE.In another work , a sulfu−1 and favorable cycling performance of 740 mAh g−1 after 200 cycles. The interaction between the rGO and sulfur not only enhanced electronic conduction in the cathode but also suppressed the volume change during the conversion reaction.Kızılaslan et al. investigated the effects of two different synthesis methods of rGO-S composites cathodes, including the melt-diffusion method and solution-based techniques . They ar2ZnSnS4/GO) via a hydrothermal approach. GO could deposit and adhere to the surface of the materials due to its hydrophilic nature as it contains more oxygen contents as compared to other forms of graphene. The results revealed a discharge capacity of 645 mAh g−1 after 50 cycles (2ZnSnS4/graphene (CZTS/graphene) with a weight ratio of 2:1 using a bilayer solid electrolyte (LGPS and 70%Li2S-29%P2S5-1%P2O5). Furthermore, a good rate of performance was observed where discharge capacities ~527, ~439, and ~278 mAh g−1 were delivered at 100, 250, and 1000 mA g−1, respectively. TEM and HRTEM images, as shown in Wan et al.  designed0 cycles c for Cu22S4, CuCo2S4/graphene, and CuCo2S4/graphene@Li7P3S11 nanocomposites were studied [2S4 aqueous solutions were mixed and dried via hydrothermal processes, resulting in composites as shown in 7P3S11 was coated on the surface of CuCo2S4/graphene nanosheets . To improve Li anode performance, the incorporation of novel nanostructured materials such as graphene with Cu is being actively considered [−2  is a preferred current collector for Li anode due to its electrochemical stability at low potentials  and natural graphite (NG) demonstrated better rate capability and capacity retention than electrodes without rGO  was created by using monolayer graphene grown on a Cu foil as the electrode (∼25 µm thick) and integrated with poly-(ethylene glycol) borate ester thin SPE [Flexible SSBs will require the development of not only flexible SSEs but also thin and flexible electrodes ,114,115.thin SPE . The pra−2 S cm−1 have been reported [+ ions in a solid-state system depends on the following two important factors: firstly, the (electro-)chemical stability at the interface; and secondly, the contact intimacy and mechanical robustness. Once Li contacts the SSE, interphase layers start to form due to Li reaction and electrolyte decomposition [+ ion transport [+ ions to form alloys that would ensure stable interfacial contact.With SSEs, the focus has been on enhancing the room temperature ionic conductivities of the electrolyte to reach or even surpass those achieved for conventional organic liquid electrolytes. There has been success on this front, especially with ceramic electrolytes where room temperature ionic conductivities >10reported ,118,119.reported . The eleposition ,120. Intransport ,122. Furransport ,125,126.+ → LiF + Gr), GF was converted to graphene and LiF that acted as an interlayer between LFP and LLZO, as shown in +. Since LLZO is very brittle in nature, achieving an intimate electrode|SSE contact is always an issue. The authors claim that a flexible graphene/LiF interlayer ensures an intimate contact that imparts stability to the Li+ ion transport; consequently, an improved battery performance (140 mAh/g after 60 cycles) is achieved than cells without interlayers (+ → 2Li+ + rGO + H2O) and subsequent coating of poly(propylene carbonate) results in the formation of an in situ rGO that acts as an interlayer. The GO-modified electrolyte showed good properties such as a wide electrochemical window (up to 4.8 V), ionic conductivity (~2 × 10–4 S cm–1), and a high ionic transference (0.9). When the interface-modified SPE was paired with Li metal and NCM622 electrodes, an initial specific capacity of about 160 mAh g−1 at 0.5 C was achieved. While there was some capacity decay, nevertheless, ~100 mAh g−1 was still maintained after 200 cycles. GO has also been used as a thin interlayer between Li and sulfide SSEs [7P3S11 (LPS) SSE from directly contacting Li metal, which otherwise would cause undesired reactions. In another work, coating GO thin layers on LiNi0.5Mn0.3Co0.2O2 (NMC532) and then integrating with a poly(propylene carbonate) based SPE helped retard the decomposition of PPC electrolyte that may be induced by the oxidized species (Ni3+ and Co4+) during the charging cycle of the cathode [Several reports demonstrate the use of graphene or GBMs at the electrode|SSE interfaces via simple facial approaches ,129,130.erlayers c. The foide SSEs . Here, t cathode . The imp+ conductive layers of LiC6, thus facilitating the uniform deposition of Li and inhibiting Li dendrite formation during long-term cycling.Graphene has also been demonstrated to help achieve better contact in a 3D architectural Li metal anode . ExistinIn this review, we have presented a summary of the recent progress in SSBs, with a focus on the role of GBMs in enhancing their electrochemical performance.Graphene exhibits novel electrochemical properties such as low charge transfer, wide potential window, excellent electrochemical activity, and fast electron transfer rate that could be useful for many applications, including LIBs. The synthesis involves chemical and/or electrochemical exfoliations of graphite to form a monolayer, FLG, or GNP graphene platelets optionally containing various functional groups. GBMs are particularly suitable since they are beneficial for each component  of an SSB as summarized in + ion transport via ordered channels provided by the 3D GO. The high mechanical strength imparted by GO has also encouraged the application of CPEs for flexible batteries. Furthermore, rGO has benefited ceramic SSEs by enhancing their fracture toughness, which could potentially be useful for inhibiting penetration of Li dendrites. It is worth mentioning that compared to less oxidized forms of GBMs, GO has various safety and regulatory hurdles that limit large-scale production. Therefore, the main applications for SSBs are mostly demonstrated at a lab-scale level.For SSEs, GO has been the most studied type so far due to its electronically insulating nature, hence excluding any possibility of electronic leakage or short-circuits, at the same time enhancing conductivities and improving mechanical and thermal properties. Ionic conductivity is improved by facilitating Li salt dissociation. Further, GOs can mechanically interlock with polymeric chains thanks to their 2D geometry, which reinforces the SPEs to achieve better mechanical properties. Lastly, SPEs based on GO can withstand higher temperatures thanks to their high thermal stability. In most of the works, the SSEs are composites of randomly oriented GOs and polymers. However, a few works have also explored the potential of using 3D GO as filler. In such structures, improved performances are observed due to enhanced LiThe role of GBMs in both cathodes and anodes is also of significant importance. Here, some of the advantages of rGO are to provide a large surface area for Li intercalation, offer high electronic conductivity to lower the electrode resistance, and suppress the agglomeration and deformation of the electrode nanostructures during cycling. In the case of the sulfur cathode, graphene can help to retard the diffusion of the polysulfides out of the cathode and suppress their shuttle towards the electrolyte and the counter electrode. Hence, not only inhibiting active material loss but also minimizing anode corrosion. They can also enhance particle-to-particle contact in transition metal oxide cathodes. On the other hand, graphene’s benefits for the anode are also substantial, such as its use as thin-film electrodes to store charge for miniature flexible thin-film batteries, with a Cu current collector to ensure less resistive Li|Cu contact, and as a template to achieve 2D morphologies for ceramic SSEs in a composite.+ ions to form alloys that would ensure an electrochemically stable and mechanically intimate electrode|SSE contact.Alongside their role in SSEs and electrodes, graphene has also the potential to be used as an interlayer at the solid–solid interface at the electrode|SSE junctions. This is mainly possible because of favorable traits of graphene such as mechanical robustness, structural flexibility, and reactivity with Li+ ion flow and minimize the risk of Li dendrites.The future is going to see an enormous demand for batteries with high storage capacity and faster charge rates. FLG and GNP with low oxygen content are becoming more commercially available and affordable and might offer a viable path once the development of graphene-enhanced SSBs gains momentum. To start with, stable SSBs should be designed that could challenge conventional LIBs in terms of performance metrics. Graphene’s role should be further explored in enhancing the electrochemical and mechanical properties of the SSEs, electrodes, and interfaces. Since GBMs have different roles for each component of an SSB, careful consideration should be given to tailoring their properties that can benefit the relevant material’s performance. GO, so far, has been the preferred material in academic research for enhancing SPEs properties. Oxidizing or altering GO’s surface chemistry by introducing new functionalized groups might improve its interaction with polymer molecules but also create more mass-production regulatory uncertainties from the health and safety standpoint. For electrodes, controlling the size and chemistry of the graphene’s micro and nanostructures will be very important. The roles of rGOs and FLG will be significant, especially in the design of electrodes. Here rGO and FLG should offer high surface area providing greater cites for Li to intercalate. They should have high electronic conductivity to help reduce cell polarisation and facilitate redox activity. Lastly, graphene, as an interface mediator, should have the mechanical flexibility to buffer the Li anode expansion and ensure a robust interfacial contact with the SSE. They should also adhere to the uneven surfaces at the electrode|electrolyte solid-solid junctions to fill up micropores that will regulate LiCurrently, the use of graphene in SSBs is still in an early stage. However, the near future is going to see an enormous demand for GBM-enhanced SSBs. To achieve the target performance, systematic research strategies should be developed, considering both performance and mass-production limitations. We believe that the pristine form of graphene is much more regulated, inexpensive, and commercially available, and therefore it is likely that more research will be performed on FLG and GNP in the near future, even though the properties of GO could be more suitable in certain cases. There will be a need for the development of more powerful in situ and operando characterization tools, which could provide insights into the interactions of GBMs with various components of an SSB. Further, computational studies as a supplement to experimental efforts may allow researchers to devise better strategies and design advanced solid-state energy storage systems.


The results showed clear differences in the composition and diversity of microbial communities among habitats, with greater dissimilarities between water column and sediment than between FL and PA communities. The microbial community assembly was dominated by dispersal limitation, ecological drift, and homogeneous selection, but their relative importance varied in different habitats. The planktonic communities were mainly shaped by dispersal limitation and ecological drift, whereas homogeneous selection played a more important role in structuring the benthic communities. Furthermore, the assembly mechanisms differed between FL and PA communities, especially in the bottom water with a greater effect of ecological drift and dispersal limitation on the FL and PA fractions, respectively. Linking assembly process to co-occurrence pattern showed that the relative contribution of deterministic processes (mainly homogeneous selection) increased under closer co-occurrence relationships. By contrast, stochastic processes exerted a higher effect when there were less inter-taxa connections. Overall, our findings demonstrate contrasting ecological processes underpinning microbial community distribution in different habitats including different lifestyles, which indicate complex microbial dynamic patterns in coastal systems with high anthropogenic perturbations.Unraveling the mechanisms structuring microbial community is a central goal in microbial ecology, but a detailed understanding of how community assembly processes relate to living habitats is still lacking. Here,  Microorganisms are highly diverse in taxonomic composition and metabolic capability, and play important roles in global biogeochemical cycles . IntensiThe marine environment provides a variety of habitats, such as waters and sediments, which promote divergent adaption by adopting different lifestyles. Regarding water columns, two types of lifestyles exist, namely free-living (FL) and particle-associated (PA). The microbial communities inhabiting these habitats are distinctly different, and are likely assembled under different mechanisms . For exaExamining the contribution of deterministic and stochastic processes in microbial community assembly can manifest the ecological strategies of coexisting species . Recentlverse sediment; FL verse PA) are assembled under different ecological processes and whether patterns of community assembly are related to species co-occurrences.Our analyses were performed in the Bohai Sea (BS), a relatively isolated coastal area in the Chinese coastline, with little exchange with other seas and a long seawater renewal time , and fluorescence (0.29–7.82 mg/m3) of the seawater samples (surface and bottom waters) were determined using a Sealogger conductivity-temperature-depth . The dissolved inorganic nutrients  were analyzed by an auto-analyzer .Seawater and surface sediment samples were collected from the Bohai Sea  of China during July–August 2019, and the location of sites are shown in TransStart FastPfu polymerase, 2 μl of 2.5 mM dNTPs, 0.8 μl of 5 μM each primer, 4 μl of TransStart 5× FastPfu buffer, and 10 ng of template DNA. The PCR amplification conditions were as follows: 95°C for 3 min, followed by 35 cycles at 95°C for 30 s, 55°C for 30 s, 72°C for 45 s, and a final extension at 72°C for 10 min. The amplified PCR products were extracted from 2% agarose gel and purified using the AxyPrep DNA Gel Extraction Kit . The purified amplicons were sequenced on the Illumina Miseq PE300 platform , performed by Majorbio Bio-Pharm Technology Co., Ltd., Shanghai, China. The raw sequencing data had been deposited in National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA) database under accession number PRJNA855144, and in the NODE database under accession number OEP003571.Total DNA was extracted from the seawater and sediment samples using the DNeasy PowerSoil Kit  according to the manufacturer’s protocols. The primer pair 515F/806R  was usedThe raw reads were firstly quality-filtered by fastp version 0.20.0  to discaAll analyses were performed in the R software version 4.13 unless otherwise indicated.To evaluate the diversity of microbial communities, alpha and beta diversity indices were calculated using the “vegan” package . Comparibray) are the two most important parameters of the framework to distinguish different ecological processes, calculated using the “picante” package  represents the probability of stochastic loss of OTUs in the community being replaced by dispersal from the metacommunity, with smaller values indicating that the community is more affected by dispersal limitation. The values of R2 and m were calculated by the “MicEco” package  and node level  were calculated. The subnetwork of different communities was extracted from the meta-community network.Meta-community co-occurrence network based on Spearman correlation coefficients was constructed using the “igraph”  and “psyProteobacteria (39.4%), Bacteroidota (19.1%), Actinobacteriota (7.3%), and Cyanobacteria (6.0%), which accounted for more than half of the community  and decreased in bottom water (3.8%) and sediment . Compared to seawater, the sediment samples were relatively enriched with Planctomycetota, Desulfobacterota, Chloroflexi, Acidobacteriota, and Nitrospirota . Abundant orders in the sediment samples included Anaerolineales (Chloroflexi), Thermoanaerobaculales (Acidobacteriota), Desulfobacterales (Desulfobacterota), and MSBL9 (Planctomycetota).Illumina sequencing generated a total of 1,373,925 clean reads, which were clustered into 15,846 OTUs at a 97% sequence similarity level. The flattening of the species accumulation curve indicated that the sequencing data were able to recover most of the local species . The seqommunity . These fnt 0.1%; . By contActinomarinaceae, Clade I (SAR11), and AEGEAN-169 marine group were more abundant in the FL fraction, whereas the PA community especially in surface water was relatively enriched by Saprospiraceae belonging to Chitinophagales in Bacteroidota, followed by Stappiaceae . No similar trend was observed in the surface water, where the average value of Shannon index appeared to be higher in FL than in PA communities though with no statistical significance  than between lifestyles ; however, an opposite trend was observed for the niche breadth values in bottom water (p < 0.001).The community-level niche breadth values varied significantly among habitats  and pH , whereas the PA community showed no significant correlations with environmental factors. In bottom water, pressure , and nitrite  correlated to the FL community, while temperature  and density  correlated to the PA community.To further investigate the underlying mechanisms that structure the microbial community in different habitats, partial Mantel test was performed to correlate distance-corrected dissimilarities of microbial community with those of environmental factors . A detaiThe |βNTI| values showed that FL and PA communities in surface and bottom waters were mainly influenced by stochastic factors, whereas the effect of deterministic processes was more important than stochastic processes in the benthic communities . Dispers2) was higher in seawater than in sediment, but the migration rates (m) exhibited an opposite trend , Planctomycetota (10.6%), Patescibacteria (10.0%), Proteobacteria (7.0%), Verrucomicrobiota (4.8%), Chloroflexi (4.5%), and Bacteroidota (3.4%) mainly occupied the nodes. The majority of edges (98.0%) in the network were positive, indicating a predominantly cooperative relationship across the microbial community.A co-occurrence network of 442 nodes and 21,188 edges was generated for the microbial community based on Spearman’s correlation coefficients . The netTo estimate the extent of species co-existence in different habitats, network topology parameters for each of the community groups were calculated . The netExploring the mechanisms that govern community assembly has been a central task in microbial ecology. In this study, we examined the assembly mechanism of FL and PA communities in seawater of the BS and compared it with that of the sediment populations. We found that the dissimilarities in community between water column and sediment were greater than those between FL and PA communities. Dispersal limitation, homogeneous selection, and ecological drift were the dominant processes in microbial community assembly but showed obvious difference among habitats. Our results suggest that ecological processes underlying the microbial community reflect the influence of environmental homogeneity and the potential influence of human disturbance, niche selection, and putative interspecies interactions.Proteobacteria, Bacteroidetes, and Cyanobacteria were found to be dominant groups and were abundant in both water and sediments. These groups have been characterized as the important groups in eutrophic coastal ecosystems  in the PA community. Chitinophagales belonged to Bacteroidetes containing members associated with algal-derived organic matter degradation  can be found at: JiwL and X-HZ designed the experiments. JinL and XW analyzed the data and wrote the manuscript. JiaL and XL carried out the laboratory experiments. JiwL, X-HZ, XW, and JinL revised the manuscript. All authors contributed to the article and approved the submitted version.R/V Beidou implementing the open research cruise NORC2019-01 supported by the NSFC Shiptime Sharing Project (no. 41849901).This work was supported by the National Natural Science Foundation of China , the Fundamental Research Funds for the Central Universities (nos. 202141009 and 202172002), and the National Key Research and Development Program of China (no. 2018YFE0124100). Data and samples were collected onboard of 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.