Arabidopsis thaliana seedlings are widely used as a model to study the de-etiolation process, the etiolation itself at the molecular level still needs elucidation. Here, we monitored the etiolation dynamics for wild type A. thaliana seedlings and lutein-deficient (lut2) mutant between 2 and 12 days of their growth in the absence of light. We analyzed the shape of the apex, the growth rate, the carotenoids and protochlorophyllide (Pchlide) accumulation, and the light-dependent protochlorophyllide oxidoreductase (LPOR) transcripts. Differences concerning the apical hook curvature and cotyledon opening among seedlings of the same age were observed, mostly after day 6 of the culture. We categorized the observed apex shapes and presented quantitatively how distribution among the categories changed during 12 days of seedling growth. The Pchlide654/Pchlide633 ratio, corresponding to the amount of the photoactive Pchlide, was the highest in the youngest seedlings, and decreased with their age. LPORA, LPORB, and LPORC transcripts were detected in etiolated seedlings, and their content decreased during seedling growth. Expression of SAG12 or SAG13 senescence markers, depletion in antioxidants, and excess ion leakage were not observed during the etiolation. Lack of lutein in the lut2 mutant resulted in slow Pchlide accumulation and affected other xanthophyll composition.Although etiolated  Light is a driving force for photosynthesis, enabling the autotrophic lifestyle of plants. Angiosperms, the most developed photosynthetic organisms, use light not only to power photosynthesis but also to manage plant growth. In the absence of light, angiosperm seedlings undergo etiolation that involves morphological, physiological, and biochemical processes that are evolutionary adaptations for efficient resource management, increasing the chance of survival . These pIn etiolated seedlings, at the cellular level, proplastids develop into etioplasts instead of chloroplasts . A paracrystalline lipid structure, called prolamellar body (PLB), is characteristic for etioplasts . The bioArabidopsis thaliana  proteins  as a model, we reveal how the lack of lutein  affects the accumulation of other pigments, in particular Pchlide, during the etiolation of seedlings. We also investigated the relative content of Pchlide: LPOR: NADPH complexes and the accumulation of LPOR transcripts as key components providing an efficient de-etiolation. In light-grown plants, the lack of lutein is compensated by increase of β,β-xanthophylls content  up to 12 days of seedling growth in darkness. We analyzed seedling morphology concerning the shape of the apex, biochemistry related to Pchlide and carotenoid accumulation, and verified whether senescence or stress is induced by the lack of light.Investigation of the dynamics of etiolation process was the other purpose of this study, because dark-grown seedlings of gulation . HoweverA. thaliana Col-0  and lut2 mutants [SALK_019364C . Parent lut2 plants used in this experiment were homozygous, confirmed by genotyping  for 7 min. Then, they were washed 6–10 times with sterile, deionized water. The seeds were sown on a plant culture plate  with a Murashige and Skooge (pH 5.7) medium , containing 1% agar (without sucrose supplementation) and irradiated with ambient white light for 2 h to improve and synchronize germination. Subsequently, plates were wrapped in a double layer of aluminum foil and placed at 4°C for 24 h (stratification). After that, seedlings were grown at 22 ± 2°C in darkness  for 2, 3, 4, 6, 8, and 12 days. Then, the seedlings were collected, gently dried with a paper towel to remove any water droplets, and weighed.Harvesting of the seedlings and all preparations of samples for analyses described below were performed under scattered dim green safe light, which was previously tested and did not induce chlorophyll biosynthesis in the seedlings see .3  for buffering. The final volume of the extract was 2 ml. The extract was centrifuged , and the supernatant was taken for pigment analysis. Total carotenoid content was determined spectrophotometrically  according to ®, Sigma-Aldrich; St. Louis, MO, United States) at room temperature. The excitation wavelength was 440 nm, and the emission in the range between 600 and 700 nm was recorded  with background subtraction. The relative Pchlide content was taken as the intensity of Pchlide fluorescence peak (at 633 nm) and normalized to seedling FW or mass of sown seeds.Carotenoid and Pchlide were extracted from weighted seedlings (about 100 mg) by homogenizing in small aliquots of 100% acetone with addition of CaCO2 (1 mM). Glass capillaries  were quickly filled with homogenate and immediately frozen in liquid nitrogen. All chemicals were purchased from Merck . Fluorescence emission spectra of A. thaliana homogenates were recorded using a steady-state spectrofluorimeter  at 77 K with excitation wavelength of 440 nm and emission, ranging from 600 to 700 nm . After background subtraction, intensities of Pchlide fluorescence peaks at 654 and 633 nm were read from the spectra and used for the calculation of the Pchlide654/Pchlide633 ratio.The cotyledons from about 50 mg of etiolated seedlings were cut off and briefly homogenized with a micropestle in a 1.5-ml centrifuge vial using 150 μl of an ice-cold 25-mM HEPES-NaOH buffer (pH 7.5), supplemented with D-sorbitol (0.4 M), EDTA (1 mM), and MgClWeighted seedlings (about 100 mg) were lyophilized in darkness, weighed again and extracted with small portions of the extraction solution containing: acetonitrile:ethyl acetate:0.2-M ammonium acetate . Aliquots of 100 μl of pigment extracts were separated using HPLC , coupled with a UV-VIS detector , using an octadecasilane column . The elution protocol is given in Antioxidants were extracted from weighted etiolated seedlings (70–100 mg) in 2 ml of ice-cold methanol . Methanolic extracts of etiolated seedlings were mixed evenly by volume with freshly prepared 1 mM ice-cold methanolic solution of 1,1-diphenyl-2-picrylhydrazyl . The spectra were measured at 20°C, using a MiniScope MS300  spectrometer with a frequency of 9.4 GHz, amplitude modulation of 1,700 mG, microwave power of 10 mW, and a scan rate of 280 G/min. First measurement was taken immediately after sample preparation, the second one after 30 min of dark incubation at room temperature. Scavenging of DPPH was calculated to quercetin  equivalent [according to SAG12 and SAG13 for which 0.33 μM was used) and amount of cDNA corresponding to 50 ng of RNA.About 50–100 mg of cotyledons was frozen in liquid nitrogen and grounded to powder. About 1 μg of total RNA extracted with Spectrum™ Plant Total RNA Kit (Sigma-Aldrich) was reverse transcribed using RevertAid First Strand cDNA Synthesis Kit and oligo(dt)18 primers. Real-time reverse transcription (qRT-PCR) was performed using SYBR Green JumpStart Taq ReadyMix (Sigma Aldrich), 0.5 μM of primers  PORB  or PORC , reference genes , SAND  and UBC , senescence markers  and SAG13 . The PCR reactions were performed using Illumina Eco™ Real-Time PCR System with Eco Control software . Reactions were initiated at 95°C for 10 min, followed by 40 cycles of 15 s at 95°C, 15 s of annealing  and 20 s at 72°C. GeNorm v3.4  were immersed with distilled water (2 ml) and incubated in darkness at room temperature for 90 min, followed by measurement of liquid conductivity (A) using Econo™ Gradient Monitor (Bio-Rad). Additionally, the conductivity of boiled (15 min at 100°C) samples was measured to estimate the maximal ion leakage (B). The index of injury was calculated using (100% × A)/B equation based on .Seedlings on culture plates were photographed immediately after removing the aluminum foil covering the plates. The images were then used for analysis of seedling morphology concerning the shape of cotyledons and apical hook curvature. A stereo microscope  equipped with an HDMI series camera and OPTAView-IS (ver. 4.3.0.6001) software was used for image acquisition and processing. Seedling lengths were calculated using ImageJ v1.52a .via two-way ANOVA for the effect of mutation and the length of the etiolation, and post hoc Tukey’s test. Differences were considered statistically significant for p < 0.05. For comparison of seedling morphology, Kruskal-Wallis with post hoc Dunn tests was used.All biochemical analyses, described in sections “Carotenoid and Pchlide determination” to “Ion leakage analysis”, were performed for 3–4 independent experiments, consisting of pooled seedling samples (the amount of seedlings is indicated in each section above). Absorption, fluorescence and EPR spectra, chromatograms, and PCR reactions were performed in triplicate. Seedling morphology was estimated using 100–300 seedlings per experiment, and the experiment was repeated three to five times. Data for plot representation were calculated as an arithmetic mean with a standard deviation (SD). Results were analyzed A. thaliana, it is not applicable for dark-grown seedlings, and no such scale has been developed for etiolated plants. Despite the vital role of A. thaliana in understanding the molecular mechanisms controlling plant etiolation, data on morphology of etiolated A. thaliana seedlings at different age are scarce. On the contrary, numerous manuscripts for cereals, Beta vulgaris, Phaseolus vulgaris, or Pisum sativum  were grown aseptically for different times between 2 and 12 days, counted from the end of stratification. The average fresh weight (FW) of seedlings collected at subsequent days of growth in darkness is shown in lut2 mutant up to the 8th day of growth. The FW of WT seedlings reached the maximum at day 6 and then decreased, while that of lut2 seedlings continued to grow till the end of analysis. The difference between the repetitions of the same experiment was ca. 20% on average. The decrease in FW observed for WT at the end of the dark cultivation period  is likely due to water loss, which does not appear to be the case for the lut2 mutant. Further investigation into water status of etiolated seedlings is required to explain this observation. However, this interesting observation may be easily overlooked in typical experiments aimed at comparison of seedlings, including various mutants, only at a certain etiolation day. On the other hand, FW is often used as a normalization factor. Our results show that such approach to biochemical analysis may involve errors resulting from the loss of seedling weight during etiolation. Data shown in In the present study, etiolated nd day of growth. At that stage, their growth was intense, and their average length almost doubled between 2nd and 4th days of development, quickly exceeding 10 mm in length  (–2 s–1) applied before the stratification substantially improved germination  . We havemination . In our mination . The strmination . These p2 mutant .A. thaliana should be perceived as a dynamic process, and growth protocols need to be taken into account when comparing results for etiolated seedlings. We advocate the opinion that etiolation methodology used for A. thaliana research should be standardized to enable comparison of results, and our work may serve as the reference. Moreover, any modifications, for example, necessary to study mutants should be clearly reported.Our results clearly demonstrated that seedling population grown in the absence of light is heterogeneous, and assumption that etiolated seedlings are identical concerning the appearance of the apex is an oversimplification. The etiolation of One may think that etiolation for up to 12 days is a highly unfavorable and detrimental phenomenon. To verify whether the observed straightening of the apical hook and the opening of cotyledons  are relaA. thaliana  seedlings seem to be protected from oxidative stresses better than the older ones. The observed decrease in antioxidant content indicates that long etiolation is somehow related with stress. However, it needs to be noted that the antioxidant pool seems not to be significantly depleted even in 12-day-old etiolated seedlings.Stress or senescence may decrease the content of antioxidants. DPPH is a stable radical commonly used for determination of antioxidant capacity of plants . The totthaliana . The totthaliana  or durinthaliana . In our thaliana , and it A. thaliana, it does not exceed 20% and typically fluctuates around 10% . Moreover, any significant age-related increase in content of SAG12 and SAG13 mRNA transcripts has not been observed, which may indicate that darkness does not induce senescence in seedlings under our experimental conditions.We also compared the expression of senescence-associated genes n leaves . SAG13 tng SAG12 . IrrespeA. thaliana seedlings, even at day 12 of etiolation, did not show obvious symptoms of neither stress nor senescence and seemed healthy.To sum up, our results clearly showed that lut2 mutant, as it was discussed in section “Seedling Growth and Morphology.” Taking into account that we have not observed differences concerning the seed weight and the germination ratio between the lut2 mutant and WT (not shown), the initial mass of seeds that were sown well correlated with the number of seeds, and, in consequence, with the number of seedlings. Thus, for comparison, we performed both the normalization of the pigment content by the mass of sown seeds , i.e., Pchlide bound to the active site of LPOR and forming ternary Pchlide: LPOR: NADPH complexes in PLBs, which are ready for Pchlide to Chlide reduction upon illumination (see 633), which is not converted to Chlide with a pulse of light. In our study, the relative amount of the photoactive Pchlide, calculated as the Pchlide654/Pchlide633 ratio, decreased with the seedling age  was observed after day 8. No significant differences between lut2 and WT were observed. That is in contrast to results obtained for Phaseolus vulgaris, where the low Pchlide657/Pchlide632 ratio was observed in 2-day-old seedlings and high in 10-day-old seedlings :LPORB transcript ratios. The average mRNA levels of LPORA were only slightly lower than LPORB ones , visible in fluorescence spectra  counterbalances this risk as decreased mortality of seedlings during their de-etiolation is observed , the repressor of glutamyl-tRNA reductase . Glutamyg amount . Howeverg amount . Pchlidelut2 seedlings irrespectively of their age. Lack of lutein is obvious for this mutant. Whereas zeaxanthin has been detected in light-grown lut2 seedlings , the zeaxanthin represents a different branch (β-carotene derivative) of the xanthophyll biosynthesis pathway  is strongly suppressed in etiolated A. thaliana seedlings  – the first enzyme of the pathway. Even though, the eedlings , caroteneedlings . Analysiassembly . However mutants . The β-c mutants . Moreove mutants .A. thaliana seedlings grown in the absence of light. They revealed that straightening of the apical hook and partial cotyledon opening occur in etiolated seedlings grown in true-dark conditions. We described heterogeneity of the morphology among seedlings in a culture. Even for a long etiolation period, seedlings do not show stress and senescence symptoms. Further research is required to elucidate molecular mechanisms of the apical hook and cotyledon opening in etiolated seedlings in true dark conditions, particularly to determine the role of hormones, PIFs factors, and COP/DET/FUS proteins.Our results show the dynamics of the development of 654/Pchlide633 ratio, which is generally considered as disadvantageous during de-etiolation, corresponds to the age-related decrease in all LPOR transcripts and to Pchlide accumulation. Lack of lutein in A. thaliana lut2 mutant has little effect on development and morphology of etiolated seedlings. However, lutein may be involved in regulation of β-xanthophylls and Pchlide biosynthesis. Further investigation is required to verify how the altered xanthophyll content and the lowered antioxidant level impact the greening of lut2 seedlings.Age-related decrease in the PchlideA. thaliana seedlings is very important for data interpretation. Moreover, to enable data comparison, etiolation methodology used for A. thaliana research should be standardized regarding the length of the etiolation time period, sugar concentration in the growth medium, and seed pretreatment with light (leading to pseudo- or true dark growth).Our work clearly showed that the way of normalizing results of pigment content in etiolated The original contributions presented in the study are included in the article/PJ and BM-K designed the experiments, analyzed the results, and worked at manuscript preparation. PJ performed the majority of experiments and coordinated the whole work. MC, PZ, and AKB planned, performed, and analyzed the qRT-PCR. KFT planned, performed, and analyzed HPLC experiments. BM-K provided funding. AKB, KFT, and PZ participated to wrote the manuscript. All authors accepted the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Rhabdomyosarcoma is the most common soft tissue sarcoma in children, and embryonal rhabdomyosarcoma is the most typical type of rhabdomyosarcoma. The heterogeneity, etiology, and origin of embryonal rhabdomyosarcoma remain unknown.After obtaining the gene expression data of every cell in the tumor tissue by single-cell RNA sequencing, we used the Seurat package in R studio for quality control, analysis, and exploration of the data. All cells are divided into tumor cells and non-tumor cells, and we chose tumor cells by marker genes. Then, we repeated the process to cluster the tumor cells and divided the subgroups by their differentially expressed genes and gene ontology/Kyoto Encyclopedia of Genes and Genomes analysis. Additionally, Monocle 2 was used for pseudo-time analysis to obtain the evolution trajectory of cells in tumor tissues.Tumor cells were divided into 5 subgroups according to their functions, which were characterized by high proliferation, sensing and adaptation to oxygen availability, enhanced epigenetic modification, enhanced nucleoside phosphonic acid metabolism, and ossification. Evolution trajectory of cells in tumor tissues is obtained.We used pseudo-time analysis to distinguish between mesenchymal stem cells and fibroblasts, proved that embryonal rhabdomyosarcoma in the pelvic originated from skeletal muscle progenitor cells, showed the evolutionary trajectory of embryonal rhabdomyosarcoma, and improved the method of evaluating the degree of malignancy of embryonal rhabdomyosarcoma. The origin of ERM is unclear, but it is currently thought to originate from skeletal muscle progenitor cells (SMPCs) or endothelial progenitor cells (EPCs), which may be related to the distribution of ERM.,3PAX7 gene expression is an essential marker of skeletal muscle differentiation, and RAS gene family are abnormally expressed in ERM. They are of particular significance in the diagnosis of ERM.Rhabdomyosarcoma is the third most prevalent extracranial solid tumor in childhood , accounting for approximately 4.5% of all childhood cancer cases. Compared with traditional DNA sequencing methods, scRNA-seq avoids interference from background cells and captures important information expressed within only a few cells, such as the mutation status of tumor cells, the epigenetic status, and the expression level of related proteins.Single-cell RNA sequencing (scRNA-seq) is a type of sequencing technology that obtains the complete gene expression information of every cell. This method can fully define the expression of transcription factors, growth factors, receptors, solute transporters, and other proteins in every cell, and it has been used in tumors to study tumor heterogeneity.In this study, we used scRNA-seq to reveal the heterogeneity in ERM and explore the evolutionary history of ERM.22.1This study included a patient with ERM in the pelvic region who underwent imaging (computed tomography and magnetic resonance imaging) and immunohistochemistry, and the patient was a 6-year-old boy with ERM in stage III. This study was approved by the ethics committee of Fudan University Children's Hospital and informed consent was obtained from the participant's guardian. All experiments were carried out in accordance with the relevant guidelines and regulations.2.2g for 8 min. The single-cell suspension was separated by human lymphocyte separation fluid , and red blood cells and cell debris were removed according to the manufacturer's specifications. The granulosa cells were resuspended in phosphate-buffered saline with 1% bovine serum albumin, and their activity and size were evaluated with a Countess II FL instrument (Thermo).The tumor tissue was cut into small pieces; collagenase IV (Gibco) and DNase I (Sigma) were added and the mixture was stirred at 37°C for 30 min. The samples were filtered through a 70-μM cell filter; the filtrate was washed in phosphate-buffered saline with 1% bovine serum albumin and 2 mM ethylenediaminetetraacetic acid and centrifuged at 500×2.3 In short, cellular suspensions were loaded on a Chromium Controller instrument (10× Genomics) to generate single-cell gel bead-in-emulsions (GEMs). After the formation of GEMs, the cells were lysed, and the GEMs were automatically dissolved to release several barcode sequences. mRNAs were reverse-transcribed to generate cDNAs with barcode and UMI information, and a cDNA library was established. The library was then pooled and sequenced on a NovaSeq 6000 (Illumina) at a depth of approximately 400M per sample. Raw sequencing data were converted to FASTQ files with Illumina bcl2fastq (version 2.19.1) and aligned to the human genome reference sequence (GRCh38). CellRanger  was used to perform data processing to filter out the barcodes associated with low-quality cell bar codes.ScRNA-seq libraries were prepared according to the Single Cell 3’Reagent Kit User Guide v2 (10× Genomics).2.4https://github.com/satijalab/seurat) was used to process and analyze the obtained gene expression data. From quality control, genes expressed in no more than 3 cells were filtered out, and cells with more than 500 genes and less than 10% mitochondrial genes were selected. Then, the remaining data were standardized and normalized. After quality control and normalization, the nonlinear dimension reduction algorithm principle component analysis was implemented. Finally, cluster analysis was used to identify cell subtypes and Uniform Manifold Approximation and Projection for visualization of dimension reduction results. A heatmap of the top 10 genes unique to every cluster was displayed according to the log2FC values.Data were imported into R Studio (R 3.6.2), and the Seurat package  to perform gene ontology (GO)/Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. First, the bitr function was used to convert the gene ID. Then, the enrichGO and enrichKEGG functions were used to perform GO/KEGG enrichment analysis of the DEGs, and GO/KEGG terms with false discover rate less than 0.01 or 0.05 were considered significantly enriched. Finally, the enrichplot package was used to visualize the enrichment results.FindAllMarkers function was used to export the differentially expressed genes (DEGs) of every cluster during Seurat analysis. After Seurat analysis, we used the clusterProfiler package (2.5http://cole-trapnell-lab.github.io/monocle-release) to conduct pseudo-time analysis in the cells. Monocle 2 used an algorithm to learn the changes in gene expression sequences that each cell must go through as part of a dynamic biological process . The cells were reduced dimensionality by the DDRTree method, sequenced in pseudo time, and finally visualized.We used Monocle 2 . Therefore, cluster 10 was composed of T cells (CD3+) and NK cells (CD3−).CLEC10A is a specific marker of human CD1C+ dendritic cells, so cluster 13 comprised dendritic cells. CD34, PROM1, and KDR are common marker genes of EPCs, so cluster 15 comprised EPCs. Cluster 7 can be labeled with CD44 and COL1A1. CD44 is a marker gene of mesenchymal stem cells (MSCs) and COL1A1 could be expressed in fibroblasts,,20 so cluster 7 might comprise MSCs or fibroblasts . Cluster 14 could be labeled with CD44, VCAM1, and TAGLN genes, which are expressed in both MSCs and fibroblasts so that Cluster14 may also comprise MSCs or fibroblasts .–24 In Section 3.3.2, we knew cluster 7 comprised cancer-associated fibroblasts (CAFs) and cluster 14 consisted of MSCs. The remaining undefined cells were tumor cells and could be defined by PAX7, HRAS, and KRAS genes.,6 The plot of ERM tumor tissue cellular composition was shown in Figure Seurat analysis was performed in all cells in the tumor tissue, and the heatmap of the top 10 marker genes for every cluster was shown in Figure 3.2To investigate the heterogeneity of tumor cells in ERM, we extracted tumor cells. Then, Seurat analysis and GO/KEGG analysis was performed for them. The heatmap of the top 10 marker genes for every cluster was shown in Figure MKI67,TOP2A, and CDK1 genes. According to GO and KEGG analysis of the DEGs in every cluster, this group of cells could be further divided into 2 subgroups. Cluster 3 was in G1 and S stages of mitosis, and the DEGs were mainly involved in the synthesis of DNA, RNA, and chromosomes, such as deoxyribonucleoside triphosphate metabolic process, DNA biosynthetic process, etc. Clusters 5, 7, and 8 were in G2 and M stages of mitosis, and the DEGs were mainly involved in the mitotic nuclear division, organelle fission, chromosome segregation, and sister chromatid segregation. Although cluster 10 was also highly proliferative, it was different from clusters 3, 5, 7, and 8. Marker genes for high proliferation could not define cluster 10, and the GO/KEGG analysis results in cluster 10 were different from other clusters too. GO/KEGG analysis showed that cluster 10 genes were mainly involved in the positive regulation of cell cycle, regulation of mitotic cell cycle phase transition, and regulation of cell cycle phase transition. The second subgroup of cells (cluster 2) could sense and adapt to oxygen availability and promote angiogenesis; this group was labeled with the VEGFA gene. GO/KEGG analysis showed that the DEGs in this group were mainly involved in response to hypoxia, decreased oxygen levels, and HIF-1 signaling pathway. The metabolism of nucleoside phosphoric acid was enhanced in the third subgroup of cells (clusters 4 and 9). GO/KEGG analysis showed that the DEGs were mainly involved in the metabolism of nucleoside phosphoric acid, ATP metabolic process, and oxidative phosphorylation. The epigenetic modification was enhanced in the fourth subgroup of cells (cluster 6). GO and KEGG analysis showed that the DEGs in this group of cells were mainly involved in protein modification, RNA splicing, chromosome tissue regulation, and other epigenetic modification processes. The fifth subgroup of cells (clusters 0 and 1) may have ossification and ossification regulation functions. GO and KEGG analysis showed that DEGs in cluster 0 were mainly involved in ossification, and those in cluster 6 were mainly involved in ossification regulation. The feature plots of the above marker genes were shown in Figure The first subgroup of cells  was highly proliferative and was labeled with 3.33.3.1TAGLNhi, Fig. Undefined cells were extracted and regrouped, and Seurat analysis was performed to divide the cells into 5 clusters Fig. B. Among 3.3.2CD44hiTAGLN-), cluster 12 comprised undefined cells-2 (TAGLNhi), cluster 13 comprised EPCs (CD34hi)  are expressed in tumor cells, but MYOG, MYOD1, and TTN (marker genes of myocytes) are rarely expressed in tumor cells (Fig.  Therefore, ERM is regarded as an arrested state in normal skeletal muscle development and is hence considered to originate from SMPCs. However, the feature plot showed that the PEG3/PW1 gene is expressed in tumor cells (Fig. PEG3/PW1 is a marker of a subset of vascular-related EPCs. Therefore, ERM in the pelvic may also originate from EPCs. Pseudo-time analysis is performed to explore the origin of ERM in the pelvic. The results show that cluster 12 comprises MSCs (TAGLNhi, Fig. lls Fig. C.34] Thlls Fig. C. PEG3/P Thlls FiTAGLN gene is expressed briefly and it is prior to tumor evolution during early skeletal muscle differentiation (Fig.  It can be used as evidence that ERM originated from SMPCs too. After that, MSCs are differentiated into CAFs and tumor cells, and the VEGFA gene is expressed in CAFs and tumor cells (Fig. VEGFA gene in pseudo-time, we speculated that the VEGFA gene is the first group of genes expressed during tumorigenesis. At the end of tumor evolution, MKI67, TOP2A, and CDK1 genes began to express (Fig.  Therefore, clusters 2, 5, 8, and 9 are mainly distributed at the end of the evolutionary trajectory, cluster 11 is distributed in the entire evolutionary trajectory. The number of highly proliferative tumor cells at the end of the evolutionary trajectory is higher than the tumor cells at the beginning. There are also some tumor cells that are expressed throughout the evolution of the tumor. There is no marker gene to define the remaining clusters, so GO/KEGG analysis is used to show the functions of every cluster. The epigenetic modification of cluster 6 is enhanced, and epigenetic modifications are altered throughout the whole process of tumor initiation and progression, so it is distributed throughout the evolutionary trajectory. The metabolism of nucleoside phosphoric acid is enhanced in clusters 4 and 10, and it is distributed at the middle of the evolutionary trajectory, this event should occur in the middle of the evolution of tumor cells. The DEGs of clusters 1and 3 are mainly involved in ossification and ossification regulation. Clusters 1and 3 are distributed throughout the evolutionary trajectory, but their role in tumor evolution is still unclear. The existing literature can only prove that ERM has an osseous component, namely, ossification can occur during the evolution of ERM. The evolutionary history of ERM is shown in Figure In the evolutionary process of ERM, ion Fig. A.37] Itlls Fig. A. Clustells Fig. A. Therefess Fig. A. These ess Fig. B. It may7 Itlls FKi-67, a marker of cell proliferation encoded by the MKI67 gene, is often used to evaluate the degree of malignancy ERM. Clinical trials have shown that the lymphatic spread and prognosis of ERM are correlated with the Ki-67 index. However, Ki-67 has some limitations as a marker of cell proliferation. The MKI67 gene is not expressed in the G1 phase, and in vitro cell culture experiments showed that Ki-67 is not needed for mammalian cell proliferation. Our results demonstrate that clusters 3 and 12 are highly proliferative but do not express the MKI67 gene (Fig. Ki-67 proliferation index. Feature plots show that TOP2A and CDK1 genes are expressed in cluster 3 (Fig. Ki-67 index may be corrected by the expression products of these 2 genes.By studying the heterogeneity in ERM, clinical diagnosis and treatment of ERM will also help. In conclusion, this paper revealed the cellular composition of ERM tumor tissue, revealed the heterogeneity in ERM, explored the evolutionary history of ERM, and improved the method of evaluating the degree of malignancy of ERM.We thank Xing-Yu Huo for editing the English text of a draft of this manuscript.Conceptualization: Bo Hong, Tian Xia, Rui Dong.Data curation: Bo Hong, Tian Xia.Formal analysis: Bo Hong, Tian Xia, Chun-Jing Ye, Yong Zhan, Ran Yang, Jia Liu, Yi Li, Zhi-Xue Chen.Funding acquisition: Rui Dong.Investigation: Bo Hong, Tian Xia.Methodology: Bo Hong, Tian Xia, Rui Dong.Project administration: Wei Yao, Kai Li, Jia Wang, Kui-Ran Dong, Rui Dong.Resources: Wei Yao, Kai Li, Jia Wang, Kui-Ran Dong, Rui Dong.Software: Bo Hong, Tian Xia, Chun-Jing Ye, Yong Zhan, Ran Yang, Jia Liu, Yi Li, Zhi-Xue Chen.Supervision: Wei Yao, Kai Li, Jia Wang, Kui-Ran Dong, Rui Dong.Validation: Bo Hong, Tian Xia.Visualization: Bo Hong, Tian Xia.Writing – original draft: Bo Hong, Tian Xia.Writing – review & editing: Wei Yao, Kai Li, Jia Wang, Kui-Ran Dong, Rui Dong.


Laodelphax striatellus is an economically important pest in Asia, especially in China. Imidacloprid, a neonicotinoid insecticide, is commonly applied in rice fields to control the planthoppers. However, the widespread application of imidacloprid also has led to the development of resistance and to other potentially negative effects on crop protection. The sublethal effects of imidacloprid have been reported in many insects. Here, we investigated the potential effects of different sublethal concentrations of imidacloprid on SBPH and found that imidacloprid could affect the fecundity, apoptosis and virus transmission in the viruliferous SBPH. The results indicated that sublethal concentrations of imidacloprid may increase the fecundity of SBPH and the impact of insecticides on the transmission of plant viruses by insects should be considered when insecticides are applied to manage insect pests.The small brown planthopper (SBPH) Laodelphax striatellus damages plants directly through sucking plant sap and indirectly as a vector of rice stripe virus (RSV), resulting in serious losses of rice yield. It is one of the most destructive insects of rice in East Asia. Insecticides are primarily used for pest management, but the sublethal concentrations of insecticides may benefit several insects. The present research attempted to explore the effects of sublethal concentrations of imidacloprid on the fecundity, apoptosis and RSV transmission in the viruliferous SBPH. The results showed that the fecundity of SBPH was significantly increased after treatment with the LC10 dose of imidacloprid, while the LC30 dose of imidacloprid reduced the fecundity compared with the control. To further investigate the underlying mechanism of increased fecundity after exposure to the LC10 dose of imidacloprid, we examined the expression levels of vitellogenin (Vg), Vg receptor (VgR) and caspases in the ovaries of SBPH, and observed the apoptosis by terminal deoxynucleotidyl transferase (TDT)-mediated dUTP-digoxigenin nick end labeling (TUNEL). qRT-PCR results indicated that the expression levels of Vg, VgR and four caspase genes were all significantly increased by the LC10 dose of imidacloprid, and TUNEL assays suggested that the frequency of apoptosis was significantly higher in the SBPH treated by the LC10 dose of imidacloprid, suggesting a potential correlation between the increased fecundity and the apoptosis of SBPH ovarioles. Additionally, the expression levels of RNA3 and capsid protein (CP) were both increased significantly by the LC10 dose of imidacloprid, whereas were decreased by the LC30 dose of imidacloprid compared to the control. Therefore, this study clarifies the mechanisms of sublethal effects of imidacloprid on viruliferous SBPH and could be used to optimize pest control strategies. Laodelphax striatellus  (Hemiptera: Delphacidae) is one of the most serious pests in East Asian rice fields, which not only causes direct damage via sucking rice sap and laying its eggs into rice tissues, but also acts as a viral vector that transmits rice stripe virus (RSV) [Vg), a precursor of vitellin in the hemolymph, attaches to the nurse cells via Vg receptor (VgR)-mediated endocytosis and then is transovarially transmitted to offspring for vertical transmission in SBPH [The small brown planthopper (SBPH), us (RSV) ,2,3. RSVus (RSV) . RSV is us (RSV) . The vir in SBPH ,6,7.The management of SBPH mainly depends on chemical insecticides, including pymetrozine, imidacloprid, buprofezin, chlorpyrifos, thiamethoxam, and nitenpyram . HoweverBemisia tabaci [Sogatella furcifera [Rhopalosiphum padi [Harmonia axyridis [Ceratomegilla undecimnotata [Aphis glycines [Myzus persicae [Vg and VgR are essential proteins related to the fecundity of females in insects, and mRNA expressions of Vg and VgR could be influenced by sublethal exposure to imidacloprid [Imidacloprid, a systemic neonicotinoid, acts as on nicotinic acetylcholine receptor via disrupting the neuronal cholinergic signal transduction, is widely used against Hemiptera insects that damage plants via sucking sap ,16. Due a tabaci , Sogatelurcifera , Rhopalohum padi , Harmoniaxyridis , and Cerimnotata . Howeverglycines  and Myzupersicae . Meanwhiacloprid ,29.caspase-Nc, caspase-8, caspase-1a and caspase-1c [Wolbachia infection, which appeared to be at least partly regulated through caspase-mediated apoptosis [B. tabaci, and the virus-induced apoptosis could increase viral accumulation and transmission in vectors [Apoptosis, a programmed cell death process, which eliminates extraneous cells in vertebrates and invertebrates, maintains the normal development and cell homeostasis to reply to external stimuli ,31,32. Tspase-1c . Much exspase-1c . Previouspase-1c ,38,39. Aspase-1c ,41 and bspase-1c ,43. Apopspase-1c . Notablypoptosis . In addi vectors .Though sublethal effects of imidacloprid have been investigated in many insects, the effects of sublethal doses of imidacloprid on the viruliferous SBPH remains unclear. In the present study, we assessed sublethal effects of imidacloprid on the fecundity, apoptosis, and RSV transmission in SBPH. These results will contribute to understanding sublethal doses of imidacloprid-mediated effects on SBPH and provide new insights on pest control.The SBPH strains were derived from a field population in Yangzhou, Jiangsu Province, China, and continuously maintained in the laboratory. Rice seedlings Wuyujing 3, supplied for SBPH, were grown in the soil at 26 ± 1 °C, 80 ± 5% RH with a 16:8 h (light: dark) photoperiod in an incubator. SBPH were fed by fresh rice seedlings (5–6 cm high) in a glass beaker .The viruliferous (RSV-infected) and non-viruliferous (RSV-free) SBPH were used to screen for RSV. When SBPH emerged about 24 h later, each pair of SBPH was mated and put into the glass tube with fresh rice seedlings. Each pair was kept for 72 h to ensure the fertilization of the female, and then the female was raised individually for oviposition. A dot immunobinding assay (DIBA)  was applThe rice seedling immersion method  was appl10, LC20, and LC30) of imidacloprid on the viruliferous SBPH, approximately 500 third instar nymphs of SBPH were transferred to the glass beakers with the rice seedlings exposed to sublethal concentrations  of imidacloprid, respectively. After 72 h exposure, the surviving insects were transferred to fresh rice seedlings for subsequent experiments.To evaluate the effects of different sublethal doses  of imidacloprid was transferred to fresh rice seedlings in glass tubes. Tap water without organic solvent and surfactant was used as a control. Each treatment contained 30 pairs of SBPH. We changed rice seedlings every two days and used the binocular stereomicroscope to count the eggs. The number of eggs were recorded until the female died. If the male died, a new male was added until the experiment ended.Approximately 24 h after emergence, each pair of SBPH exposed to different sublethal doses  at −70 °C after dissection, and Tissuelyser II  was applied to homogenize the samples to extract total RNA according to the manufacturer’s protocols. The NanoDrop 2000 spectrophotometer  was used to measure the RNA concentrations and purity.http://bioinfo.ut.ee/primer3-0.4.0/, accessed on 15 November 2021) was performed to design the primers for qRT-PCR , with the final volume of 20 µL, containing 2 μL of template cDNA, 10 μL of ChamQTM SYBR qPCR Master Mix , 0.8 μL of each primer (10 μM) and 6.4 μL of ddH2O. The procedure of qPCR was 50 °C for 2 min, 95 °C for 5 min, followed by 40 cycles of 95 °C for 15 s, 60 °C for 45 s with the step of melt curve dissociation. The relative expression level of triplicate samples was calculated using the 2−ΔΔCT method [β-actin [PrimeScript™ RT Master Mix  was used to transcribe the total RNA (1 μg) into cDNA for qRT-PCR analysis, and the Primer 3 . For apoptotic cell death analyses, ovaries of SBPH fed on rice seedlings treated with tap water and LC10 of imidacloprid were dissected. After hybridization, the samples were washed two times and then fixed on a glass slide using the DAPI-containing mounting Vectashield . Finally, an ultra-high resolution laser scanning microscope  was applied to analyze the samples. Cells stained with TUNEL and DAPI fluorescences were regarded as apoptotic cells.The greatest fecundity of SBPH appears at 4 days post-emergence, while after 10 days almost all reproductive ability is lost . TherefoDue to the difficulty of maintaining integrity of ovaries under staining, we counted the TUNEL-positive tropharia and expressed them as a percentage of the examined tropharia. The frequency of apoptotic tropharia was compared between the control and treated females.10 to LC30 values with 95% confidence limits. GraphPad Prism version 8.0.0  was used to analyze and visualize the data. The data of vertical transmission rates and TUNEL-positive rates were analyzed by a Chi-squared (χ2) test. The differences between the two groups were compared using the Student’s t-test.The probabilistic unit (Probit) regression in SPSS version 16.0 software  was used to determine the bioassay results of imidacloprid, including LC10 dose of imidacloprid, the fecundity of viruliferous SBPH was significantly increased compared to the control, while the fecundity of SBPH treated by the LC30 dose of imidacloprid was significantly lower than the control, and LC20 had no obvious effect  was significantly higher than the control at 4 days after emergence (31.5%) (p = 0.5143). The results indicated that exposure to imidacloprid increased the number of nurse cells undergoing apoptosis.Apoptotic nurse cells in the tropharium were observed in both control and treated ovaries from viruliferous SBPH at 4 and 10 days after emergence, but the number of apoptotic cells was significantly more in SBPH females treated by imidacloprid than the control . The per (31.5%) . The per10 of imidacloprid, but not significantly affected by LC20 and LC30 of imidacloprid compared with the control , compared with the control (87.1%)  A. Additiacloprid B, and thectively C. LC20 oImidacloprid, a neonicotinoid insecticide, is one of the most widely applied insecticides , particu30 dose of imidacloprid, the fecundity of SBPH was significantly reduced compared to the control. In contrast, the LC10 dose of imidacloprid stimulated the reproduction of SBPH. These results are known as hormesis, which is a biphasic dose-response characterized by high-dose inhibition and low-dose stimulation during or following exposure to toxic [M. persicae [A. glycines [Podisus maculiventris [Frankliniella occidentalis [Cyrtorhinus lividipennis [Tryporyza incertulas [Reproduction-related characters are the most essential sublethal parameter studied in the pesticide toxicology of arthropods . Our stuto toxic . This phpersicae ,54, A. gglycines , and Podiventris . In Frandentalis , Cyrtorhdipennis ,56, and certulas , the feccertulas , triazopcertulas  could alVg and VgR, and found that Vg mRNA expression was significantly up-regulated by the LC10 dose of imidacloprid and suppressed by the LC30 dose of imidacloprid, which was consistent with the fecundity of SBPH mediated by imidacloprid. Moreover, VgR mRNA expression was also significantly increased by the LC10 dose of imidacloprid. Previous studies showed that the transcript level of Vg and VgR was up-regulated in insects exposed to sublethal doses of insecticides. In SBPH, Vg and VgR mRNA expressions were increased in the females exposed to sublethal doses of triazophos [Nilaparvata lugens, the transcript level of Vg was significantly increased by low doses of deltamethrin and triazophos [C. lividipennis, Vg mRNA expression was significantly up-regulated by sublethal doses of triazophos, deltamethrin and imidacloprid [Vg and VgR mRNA expressions partly reflect reproductive changes induced by the sublethal concentrations of insecticides.We examined the mRNA expressions of iazophos . In Nilaiazophos . In C. lacloprid . These r10 of imidacloprid. Meanwhile, we found that four caspase genes were significantly increased by LC10 of imidacloprid, which were generally consistent with the increased fecundity, Vg and VgR mRNA expressions by the LC10 dose of imidacloprid. Similar results appeared in the Wolbachia-infected SBPH, suggesting that the fecundity of SBPH may be enhanced by increasing the frequency of caspase-dependent apoptosis in the ovaries of infected SBPH [The previous study showed that sublethal concentrations of imidacloprid induced the caspase-dependent apoptotic pathway in the honey bees via increasing the expression level of caspase-1 and activating caspase-3 . TUNEL sted SBPH . Our finTYLCV) transmitted by B. tabaci. For instance, sulfoxaflor significantly eliminated the transmission of TYLCV [TYLCV transmission in B. tabaci [BQCV) titers in the honey bees [10 dose of imidacloprid, but no significant differences occurred. However, a recent study has indicated that the vertical transmission rates of RSV were significantly induced by sublethal doses of triazophos in SBPH [CP gene or viral RNA3 segment is used to reflect viral load and has been widely applied to quantify RSV accumulations in SBPH [RNA3 and CP, and they were both up-regulated significantly by the LC10 dose of imidacloprid, whereas decreased by the LC30 dose of imidacloprid. These results are generally consistent with the recent study which showed that after treatment with LC20 to LC50 of triazophos, the transcript levels of RNA3 and CP were significantly stimulated in the RSV-carrying SBPH [The virus transmission in insects is closely related to the change of external conditions, including pesticides. The studies demonstrated that pesticides may inhibit the transmission of plant viruses in insects, especially the tomato yellow leaf curl virus (of TYLCV , and let. tabaci ,62. Howe. tabaci . Sublethney bees ,65,66. O in SBPH . The mRN in SBPH . Thus, oVg and VgR mRNA expressions and the increase of apoptotic cells in ovarioles. Additionally, we found that the LC10 dose of imidacloprid stimulated the replication of RSV in SBPH, suggesting that the impact of insecticides on the transmission of viruses by insects should be considered. This study will provide a theoretical basis for understanding the sublethal effects of insecticides and new insights for integrated pest management.In conclusion, this study indicated that a sublethal dose of imidacloprid could stimulate the fecundity of SBPH, which may be related to the


In the present study, a simple and environmentally friendly extraction method based on natural deep eutectic solvents (NADESs) was established to extract four bioactive steroidal saponins from Dioscoreae Nipponicae Rhizoma (DNR). A total of twenty-one types of choline chloride, betaine, and L-proline based NADESs were tailored, and the NADES composed of 1:1 molar ratio of choline chloride and malonic acid showed the best extraction efficiency for the four steroidal saponins compared with other NADESs. Then, the extraction parameters for extraction of steroidal saponins by selected tailor-made NADES were optimized using response surface methodology and the optimal extraction conditions are extraction time, 23.5 min; liquid–solid ratio, 57.5 mL/g; and water content, 54%. The microstructure of the DNR powder before and after ultrasonic extraction by conventional solvents (water and methanol) and the selected NADES were observed using field emission scanning electron microscope. In addition, the four steroidal saponins were recovered from NADESs by D101 macroporous resin with a satisfactory recovery yield between 67.27% and 79.90%. The present research demonstrates that NADESs are a suitable green media for the extraction of the bioactive steroidal saponins from DNR, and have a great potential as possible alternatives to organic solvents for efficiently extracting bioactive compounds from natural products. Dioscorea nipponica Makino, is widely distributed in China, South Korea, and Russia, and has been used as a traditional herbal medicine for relieving cough and asthma, eliminating rheumatic aches, alleviating pain, and improving blood circulation [Dioscoreae Nipponicae Rhizoma (DNR), the dried rhizome of culation . Modern culation ,3,4,5,6.culation ,8. Thereculation ,10. HoweConventional organic solvents, such as methanol, ethyl acetate, acetone, and chloroform, are commonly used in the extraction of bioactive components from natural sources. However, the consumption of large amounts of these volatile and hazardous organic solvents may contribute to environmental pollution and leave unacceptable solvent residues in extracts. Green extraction methods which are environmentally friendly and sustainable for sample preparation have received more and more attention in past years .Since being introduced as a new type of green solvents, deep eutectic solvents (DESs) have rapidly gained great interest as sustainable alternatives to hazardous organic solvents . DESs arl-proline (Pro) based NADESs were tailored for ultrasonic extraction of four bioactive steroidal saponins, including protodioscin, protogracillin, pseudoprotodioscin, and pseudoprotogracillin from DNR. Then, the extraction conditions for the four steroidal saponins, including the water content of NADESs, extraction time, and liquid–solid ratio were systematically optimized using response surface methodology (RSM), and the microstructure of the raw and extracted DNR powder were observed by field emission scanning electron microscope. Moreover, the recovery of the four steroidal saponins from tailored NADESs was tentatively carried out by D101 macroporous resins.In the present study, in order to evaluate NADESs for the extraction of steroidal saponins, a series of choline chloride (ChCl), betaine (Bet), and In order to achieve a rapid and efficient analysis of the four bioactive steroidal saponins  in DNR, several HPLC conditions, including mobile phases , flow rates , and column temperatures  were compared and optimized. As a result, the water–acetonitrile system at 30 °C with a flow rate of 1.0 mL/min was selected for the suitable analysis duration, greater separation ability, and better peak shapes of the four analytes. The chromatographic peaks of: protodioscin (1), protogracillin (2), pseudoprotodioscin (3), and pseudoprotogracillin (4) were confirmed by comparing their retention times with the reference standards. The typical HPLC chromatograms of DNR sample and the four steroidal saponins reference standards are shown in r2 > 0.9992) over the tested range. The LODs and LOQs of the four analytes were defined by the concentration that generated peaks with signal-to-noise values of 3 and 10 using standard solutions. The precision of the method was determined by the intra- and inter-day variations. For intra-day tests, the same sample was analyzed six times within the same day, while for inter-day tests, the sample was examined in duplicates for three consecutive days. The relative standard deviations (RSDs) of intra- and inter-day precisions were less than 1.4% and 2.7%, respectively. For the repeatability test, six replicates of the same sample were prepared and analyzed, and for the stability test, the same sample was stored at room temperature and analyzed by replicate injection analysis at 0, 2, 4, 8, 12, and 24 h. The repeatability presented as RSDs were less than 2.1%, and the stability were less than 3.0%. The recovery test was used to evaluate the accuracy of the method. A known amount of the four steroidal saponins reference standard solutions were added into the same samples in sextuplicate, and then the samples were extracted and analyzed with the same procedures as described in Method validation of quantitative analysis of the four bioactive steroidal saponins in DNR was performed. The linearity, limit of detections (LODs), limit of quantifications (LOQs), precision, repeatability, stability, and accuracy were validated. The calibration curves of the four steroidal saponins were performed with six different concentrations of standard solutions in triplicate. All calibration curves were of good linearity with high correlation coefficient (L-PROLINE) to prepare NADESs. A total of thirty combinations of NADESs were initially prepared, and twenty-one of them were eventually found to be stable as a clear, viscous liquid without precipitation over the course of time  were used in combinations with three types of HBAs  with water to decrease viscosities for enhancing the extraction efficiency.Compared with conventional extraction solvents, the high viscosity of NADESs restricted their application in extraction. High viscosity may impair the efficiency of extraction as slow mass transfer occurs, and the low viscosity of the NADESs led to high diffusivity and hence improved the extraction performance . In the Extraction yields of the four steroidal saponins  and the total four steroidal saponins with different types of NADESs are shown in To get insight into the NADESs’ excellence in the extraction of bioactive steroidal saponins from DNR, the extraction yields of different NADESs and conventional efficient solvents (methanol and water) were compared. For protodioscin and protogracillin, almost all the NADESs exhibited lower extraction efficiency compared with methanol, and the ChCl-Eg, ChCl-Lac, ChCl-Mal, ChCl-Mu, Bet-Lac, and Bet-Mal exhibited higher extraction efficiency compared with water. For pseudoprotodioscin and pseudoprotogracillin, the ChCl-Maa, ChCl-Mal, Pro-Xyl, and Pro-Ox exhibited higher extraction efficiency compared with methanol and water. For the total four steroidal saponins, the extraction efficiency of ChCl-Eg, ChCl-Lac, ChCl-Mal, ChCl-Mu, ChCl-Am, Bet-Lac, and Bet-Mal was higher than water with the concentration 59.20 ± 0.02, 64.27 ± 0.03, 64.99 ± 0.02, 61.36 ± 0.01, 55.31 ± 0.03, 60.05 ± 0.02, and 57.64 ± 0.01 mg/g, respectively. Compared to methanol, most NADESs showed lower extraction yields of the total four steroidal saponins, but ChCl-Lac (64.27 ± 0.03 mg/g) and ChCl-Mal (64.99 ± 0.02 mg/g) produced approximate extraction efficiency. Thus, ChCl-Mal was selected as the best NADES for extraction of steroidal saponins from DNR and applied in further tests based on the initial screening results.The molar ratio of HBAs and HBDs has been regarded as an important factor that influences the surface tension and polarity of NADESs , which cRSM was an effective method to design and analyze tests with independent variables upon different levels to optimize the results, and had been widely used in optimization of extraction processes ,26. The The variables and response were processed to build a mathematical regression model for steroidal saponins. The model was expressed as second order polynomial quadratic equation for the extraction yields (Y) and coded variables  as follows:2) and the Lack of Fit by the analysis of variance (ANOVA) at the 95% confidence level. As shown in 2 value was 0.9153, indicating that the experimental data were in relatively good agreement with predicted extraction yields. The Lack of Fit value was insignificant for the response with p-value of 0.4976 (p > 0.05), which indicated that the regression model fitted the models well and could suitably explain the data.The quality of regression model for steroidal saponins was evaluated in terms of the square of correlation coefficient (Rp < 0.05). Based on the regression model, the optimum condition of ChCl-Mal for extraction of four bioactive steroidal saponins from DNR was as following: water content in NADESs, 39.69%; extraction time, 19.98 min; liquid–solid ratios, 32.28 mL/g. For the convenience of practical operation, the optimum condition was set at water content in NADESs, 40%; extraction time, 20 min; liquid–solid ratios, 32 mL/g. Triplicate experiments were carried out under the optimal extraction conditions and mean values of experimental results were compared with the predicted values. Under the optimum conditions, the actual extraction yield of the total four steroidal saponins was 66.82 mg/g, which was closed to the predicted extraction yield of 66.89 mg/g. This results indicated that the model was credible for optimization of extraction parameters of steroidal saponins from DNR.Statistical analysis and 3D response plots  obviouslSeveral previous studies have been reported that the NADESs could replace the organic solvents as extraction media for extraction of bioactive compounds from natural products due to their great extraction performance, which agrees with the results of this study ,28. HoweIn the present study, the microstructures of the DNR powder before and after ultrasonic extraction in water, methanol, and the selected NADES  were observed by field emission scanning electron microscope. As shown in The recovery of extracted bioactive compounds from NADESs is a challenge due to the high water miscibility and non-volatilization property of NADESs. The D101 macroporous resin is a common, accessible, and inexpensive packing material for column chromatography. In this study, the recovery of the four bioactive steroidal saponins from ChCl-Mal was tentatively applied by using D101 macroporous resin column chromatography according to our previous research . As descDNR samples were purchased from a local Traditional Chinese Medicine market . The samples were identified by Associate Professor Long Guo, and the voucher specimens have been deposited in Hebei University of Chinese Medicine, Shijiazhuang, China. The DNR samples were dried in the shade and powered by a disintegrator, then sieved through 60 mesh and stored in the desiccator before use.Choline chloride (ChCl), betaine (Bet), L-proline (Pro), ethylene glycol (Eg), glycerol (Gly), xylitol (Xyl), D-sorbitol (Sor), lactic acid (Lac), malonic acid , DL-malic acid (Maa), Oxalic acid (Ox), Citrate acid (Ca), l-Methylurea (Mu), Acetamide (Am), and Urea (Ur) were purchased from Aladdin Biochemical Technology . The reference compounds of protodioscin, protogracillin, pseudoprotodioscin, and pseudoprotogracillin (purities > 95%) were obtained from Chengdu Biopurify Phytochemicals Company . The chemical structures of the four steroidal saponins are shown in NADESs can be synthesized by a heating method according to the previous study . BrieflyFor initial screening of NADESs for extraction of the four bioactive steroidal saponins from DNR, an accurately weighed DNR powder (50 mg) was extracted with 1 mL of prepared NADESs containing 30% water in a 2-mL centrifuge tube by ultrasonic  for 20 min at room temperature. After extraction, the mixed suspension was centrifuged at 13,000 rpm/min for 10 min. Then, the supernatant was diluted eight times using 50% methanol and filtered through membrane filters (0.22 μm) prior to HPLC analysis. Each extraction was performed in triplicate.The reference compounds of protodioscin, protogracillin, pseudoprotodioscin, and pseudoprotogracillin were accurately weighed and dissolved in DES , and then diluted eight times with 50% methanol to the concentrations of 840, 490, 640, and 320 μg/mL, respectively. The standard solutions containing four reference standards were prepared by appropriate dilution to a series of proper concentrations. The standard solutions were stored at 4 °C for further HPLC analysis.The concentrations of four steroidal saponins extracted by NADESs were determined using an Agilent 1260 HPLC system  equipped with a quaternary pump, an autosampler, a thermostatic column compartment and a diode array detector. Separation was performed on an Agilent ZORBAX Eclipse Plus C18 column . The mobile phase consisted of water (A) and acetonitrile (B), and the gradient conditions were as follows: 0–5 min, 15% B; 5–30 min, 15–40% B; and 30–50 min, 40–50% B. The column temperature was set at 30 °C, and the flow rate was set at 1.0 mL/min. The detection wavelength was 203 nm, and the injection volume was 20 μL.The extraction parameters of the bioactive steroidal saponins by the selected NADES were optimized using RSM, which is a statistical technique for studying the interactions between factors in an appropriate variable range. On the basis of preliminary single-factor experiments, the water content (%) in NADESs (A), extraction time (B), and liquid–solid ratios (C) were selected as three independent variables to optimize the extraction parameters by Box–Behnken design (BBD). The total extraction yields of the four bioactive steroidal saponins were taken as the response of the design experiments. Regression analysis was performed according to the experimental data, and additional confirmation experiments were conducted to confirm the validity of the statistical experimental strategies. The Design-Expert Ver. 8.0.6 software  was used for the generation and evaluation of the experimental design.A JEOL  JSM-6390A field emission scanning electron microscope in the secondary electron mode with 15-kV accelerating voltage was used for observing the microstructure of the raw and extracted DNR powder. Before imaging, the samples were coated with a Pt layer of approximately 10-nm thickness.Recovery of the four steroidal saponins from the NADESs was conducted based on microporous resin column chromatography. A glass column filled with 10 g D101 macroporous resin was used and the bed volume (BV) was 20 mL. The NADESs solution (1 mL) was loaded at the flow rate of 3 BV/h. After the target compounds have been adsorbed in the resin, the column was washed with deionized water (100 mL) first, and then eluted with 100 mL ethanol. Then, the ethanolic fraction was concentrated under vacuum, and analyzed by HPLC as described in In the present study, a green and efficient extraction method using NADESs as the extraction solvent was established for extraction of bioactive steroidal saponins from DNR. A total of twenty-one types of NADESs were successfully synthesized and used to extract four bioactive steroidal saponins, including protodioscin, protogracillin, pseudoprotodioscin, and pseudoprotogracillin from DNR. The results indicated that the tailor-made NADESs were efficient solvents for the extraction of steroidal saponins, and the ChCl-Mal with a molar ratio of 1:1 proved to be the most efficient NADES. Then, the extraction parameters of ChCl-Mal for extraction of the four bioactive steroidal saponins were optimized using RSM and the optimal extraction condition was water content in NADESs, 40%; extraction time, 20 min; and liquid–solid ratios, 32 mL/g. Moreover, the microstructure of the DNR powder before and after ultrasonic extraction were observed by field emission scanning electron microscope. The recovery of the extracted steroidal saponins from NADESs was using D101 macroporous resin and the recovery yields of protodioscin, protogracillin, pseudoprotodioscin, and pseudoprotogracillin were 79.90%, 68.12%, 67.27%, and 74.8%, respectively. The present research suggests that NADESs are efficient solvents which could be used as green media for extraction of bioactive steroidal saponins from DNR, and the data acquired in this study might contribute to further NADESs application in extraction of bioactive compounds from natural products.


Background: Chinese indigenous sheep can be classified into two types according to their tail morphology: fat-rumped and thin-tailed sheep, of which the typical breeds are Altay sheep and Tibetan sheep, respectively.Methods: To identify the differentially expressed proteins (DEPs) underlying the phenotypic differences between tail types, we used isobaric tags for relative and absolute quantification (iTRAQ) combined with multi-dimensional liquid chromatography tandem-mass spectrometry (LC-MS/MS) technology to detect candidate proteins. We then subjected these to a database search and identified the DEPs. Finally, bioinformatics technology was used to carry out Gene Ontology (GO) functional and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses.Results: A total of 3,248 proteins were identified, of which 44 were up-regulated and 40 were down-regulated DEPs. Analyzing their GO function terms and KEGG pathways revealed that the functions of these DEPs are mainly binding, catalytic activity, structural molecule activity, molecular function regulator, and transporter activity. Among the genes encoding the DEPs, APOA2, GALK1, ADIPOQ, and NDUFS4 are associated with fat formation and metabolism.Conclusion: The APOA2, GALK1, ADIPOQ, and NDUFS4 genes may be involved in the deposition of fat in the tail of sheep. This study provides a scientific basis for the breeding of thin-tailed sheep. To enable their adaptation to various environments, long-term natural selection has produced sheep breeds differing starkly in their individual phenotypes. The difference in tail type is one of the main changes in the evolutionary process of sheep. Studies have shown that fat-tailed sheep emerged 5,000 years ago, having gradually formed after the long-tailed Asian thin-tailed sheep were first domesticated . Howevervia high-resolution mass spectrometry; it is a high-throughput screening technique commonly used in quantitative proteomics in recent years (Isobaric tags for relative and absolute quantification (iTRAQ) are a proteome quantification technology with high throughput and high sensitivity. This technology uses a variety of isotope reagents to label the N-terminus of protein polypeptides or lysine side chain groups and simultaneously analyzes the protein expression between two and eight samples nt years . For exaThree Altay sheep were randomly selected from Altay in Xinjiang Province and three Tibetan sheep from Tianzhu in Gansu Province . The tai18 cartridge was used to desalt the enzymatic peptides. After the peptides were lyophilized, 40 μl of dissolution was added to reconstitute them, and the peptide D280 nm value finally measured.Protein extraction was done using the SDT  cleavage method , with thFrom each sample, 100 μg peptide was taken and the instructions of the AB SCIEX iTRAQ labeling kit followed accordingly, then the labeled peptides mixed.2PO4 and 25% ACN, at pH 3.0, and buffer B of 10 cM KH2PO4, 500 mM KCL, and 25% ACN, at pH 3.0. The chromatographic column was equilibrated with liquid A, a given sample loaded onto the chromatographic column by the injector, using a flow rate set to 1 ml/min. The liquid phase gradient went was follows: 0% B liquid for 25 min; linear gradient of B liquid from 0 to 10%, for 25–32 min; linear gradient of liquid B from 10 to 20%, for 32–42 min; linear gradient of liquid B from 20 to 45%, for 42–47 min, linear gradient of liquid B from 45 to 100%, for 47–52 min; and lastly, for 52–60 min, liquid B was maintained at 100%. After 60 min, the B liquid was reset to 0%. Absorbance at 214 nm was monitored during the elution process, and eluted components were collected every 1 min, freeze-dried, and desalted by using the C18 cartridge.Each group of labeled peptides was mixed, with the AKTA Purifier 100 (AKTA avant25 Cytiva) used for classification. Buffer A consisted of 10 cM KH6, the maximum IT was 50 ms, and the dynamic exclusion time is 60 s. The mass-to-charge ratios of peptides and peptide fragments were collected accordingly as follows: 20 fragment spectra were collected after each full scan; the MS2-activation type was HCD; the isolation window was 2 m/z; the secondary mass spectrometry resolution was 17,500, at 200 m/z; normalized collision energy was 30 eV; and the underfill ratio was 0.1%.Each graded sample was separated by the high-performance liquid chromatography (HPLC) liquid phase system Easy nLC by using a nanoliter flow rate. Buffer A consisted of 0.1% formic acid in water, and B was 0.1% formic acid-acetonitrile (acetonitrile: 84%). The chromatographic column was equilibrated with 95% liquid A. The sample was loaded onto the loading column by the autosampler, separated by the analytical column, and the flow rate was set to 300 nl/min. After the sample was separated by chromatography, mass spectrometry was performed by a Q-Exactive mass spectrometer. The detection method applied was “formal ion,” the scanning range of precursor ion was 300–1,800 m/z, the resolution of primary mass spectrometry was 70,000 at 200 m/z, the AGC target was 1eP < 0.01 with an fold-change (FC) ≥ 2 or ≤ 0.85  annotation of the target protein collection. This process includes sequence alignment, GO mapping, GO annotation, and InterProScan annotation augmentation. The KAAS [Kyoto Encyclopedia of Genes and Genomes (KEGG) Automatic Annotation Server] software was used to annotate the KEGG pathways of the target protein collection against the KEGG database.First, the quantitative information of the target protein collection was normalized [normalized to  interval]. Next, we used the “Complexheatmap” package for the R software platform (v3.4) to simultaneously categorize the two dimensions of sample and protein expression , from which a hierarchical clustering heat map was generated.2 or STRING3 database, the target protein or an indirect interaction relationship was searched for. Cytoscape software (v3.2.1) was used to generate an interaction network and analyze it.Based on the information available in the IntActFor this assay, 50–100 mg tail fat tissue was placed in an Eppendorf tube and shredded. Then, 1 ml of the tissue cell lysate was put on ice for 3 h and then centrifuged at 12,000 rpm, at 4°C for 10 min, whose supernatant fluid was collected for use in another new EP tube. A total protein sample of about 10 μl was taken, to which 10 μl of loading buffer was added, and then boiled 3–5 min to denature the protein. Next, SDS-PAGE gel electrophoresis was conducted, and protein transferred to a PVDF membrane. Finally, according to the antigen–antibody reaction, a western blot analysis of the target protein was carried out.By comparing the information obtained by iTRAQ proteomics technology and the database results, 3,248 proteins were identified from 20,120 peptides by searching against the Uniprot_Ovis_aries_27827_20180612 database. According to their segment length distribution, these peptides mainly consisted of 7–21 amino acids , a reasoP-value from the t-test.Between the fat-rumped and thin-tailed sheep, 44 up-regulated and 40 down-regulated proteins were identified, giving a total of 84 differentially expressed proteins (DEPs). A volcano graph  was drawP < 0.05 for the t-test, the DEPs obtained by the screening can effectively separate the comparison components. Accordingly, this supports the screening rationale for DEPs we employed.In this study, the hierarchical clustering algorithm was used to perform cluster analysis on the DEPs of the comparison group. These results were displayed in the form of a heat map. As Among the corresponding differentially expressed genes (DEGs) for the proteins, we found some genes related to fat synthesis and metabolism .4 software  plays an important role in fat metabolism. In recent years, the APOA2 gene has been used often as a candidate gene for fat deposition, and its genetic polymorphism distribution among different poultry was studied to find genetic markers related to production traits to inform and guide breeding production. A research by APOA2 gene is a also a key gene for fat deposition in pigs. This gene was also identified in our study, so it could also be involved in fat deposition that occurs in the tail of sheep. The NDUFS4 gene encodes the respiratory chain NADH dehydrogenase in the mitochondrial membrane, and previous studies have shown that the mitochondria play a key role in adipocyte differentiation. NDUFS4 mRNA expression in the longest back (posterior) muscle was significantly higher in the high-intramuscular fat (IMF) than low-IMF Laiwu pig, and its expression in the IMF-rich Laiwu pig also significantly exceeded that of the IMF-poor large white pig, prompting the authors to speculate that NDUFS4 affects the deposition of IMF (ADIPOQ) is the first adipose tissue found in mice, by ADIPOQ plays an important role in regulating fat metabolism, mainly by binding to receptors and acting on target tissues to exert biological effects. Animal breeds, in having different growth periods and tissue parts, as well as physiological conditions, are expected to influence how ADIPOQ regulates lipid metabolism, which in turn affects fat deposition in animals.The protein interaction network is constituted by the interaction of proteins to participate in all aspects of life processes such as biological signal transmission, gene expression regulation, energy and material metabolism, and cell cycle regulation. A systematic analysis of the interactions of a large number of proteins in biological systems is of great significance for understanding the working principles of proteins in biological systems, understanding the reaction mechanism of energy material metabolism, and understanding the functional connections between proteins. In mammals, fatty acid metabolism is intricate, and some key proteins play a vital role . In the n of IMF . AdiponeAPOA2, GALK1, ADIPOQ, and NDUFS4, could be involved in the regulation of fat deposition in the tail of sheep.The iTRAQ technology was used to compare the fat proteins in the tail of Altay sheep and Tibetan sheep, and the DEPs obtained were analyzed. It is speculated that certain genes, namely, The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE  partner The animal study was reviewed and approved by the Gansu Agricultural University , approval no. GSAU-AEW-2017-0003.CZ conceived and designed the experiments, performed the experiments, and wrote the manuscript. YM, NL, TL, and HC analyzed the data and contributed reagents, materials, and analysis tools. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


To evaluate International Endometrial Tumor Analysis (IETA) ultrasonographic characteristics simple scoring method and tumor biomarkers for the diagnosis of uterine cavity and endometrial lesions.We classified and scored the normalized description of IETA ultrasonic characteristics, according to IETA expert consensus literature, previous IETA-related research articles, and the previous research experience of this project group. We conducted a retrospective analysis of the ultrasound images of 594 patients enrolled from January 2017 to June 2020, scored them item by item, and finally calculated the total score of each case. Meanwhile, we combined the results of seven tumor biomarkers. The objective was to evaluate the sensitivity, specificity, coincidence rate, and the area under receiver operating characteristic (ROC) curve of IETA ultrasonographic characteristics simple scoring method and tumor biomarkers for benign and malignant uterine cavity or endometrial lesions. The diagnostic efficiency between the combined method and the single method was compared.p ≤ 0.01). The other five tumor biomarkers  showed no statistically significant difference in benign and malignant lesions. If the value of CA19-9 ≥13.96 U/ml was taken as cutoff value, the sensitivity, specificity, and coincidence rate of the diagnosis of endometrial benign and malignant lesions were 54.8%, 74.7%, and 70.7%, respectively, and the AUC was 0.620. If the value of HE4 ≥ 39.075 pmol/L was taken as cutoff point, the sensitivity, specificity, coincidence rate, and AUC were 77.4%, 67.9%, 69.8%, and 0.796, respectively. The sensitivity was increased to 97.6% and the AUC was 0.939 when IETA ultrasound characteristics simple scoring method combined CA19-9 and HE4 in parallel test.A total of 594 cases were confirmed by postoperative pathology or surgery records, including 475 benign lesions and 119 malignant lesions. In the simple ultrasound scoring method, the average score of benign lesions was 3.879 ± 1.279 and that of malignant lesions was 9.676 ± 4.491. If ≥6.5 points was taken as the cutoff value for the judgment of malignant lesions, the sensitivity, specificity, coincidence rate, and the area under receiver operating characteristic (ROC) curve (AUC) were 76.5%, 96.0%, 92.1%, and 0.935, respectively. The difference in tumor antigen 19-9 (CA19-9) and human epididymal protein 4 (HE4) between benign and malignant lesions was statistically significant (all In IETA ultrasound characteristics simple scoring method, with ≥6.5 points as the cutoff value, it could quickly and accurately assess the benign and malignant in uterine cavity and endometrial lesions, with high diagnostic value. The diagnostic efficacy of seven tumor biomarkers was all mediocre. Combining with these two methods, the comprehensive diagnosis could improve sensitivity and accuracy and reduce the risk of missed diagnosis. Endometrial lesions are common gynecological diseases, which have a great impact on women’s physical and mental health and quality of life. In particular, endometrial cancer is one of the most common gynecological malignancies –5 and acWomen with early-stage endometrial cancer often have no special clinical symptoms. More detailed uterine examination could make an early diagnosis, which is of great significance to the prognosis, improving the cure and survival rates of the patients. It could relieve the suffering of patients.The traditional diagnostic methods are diagnostic curettage or hysteroscopy, but both are invasive and cannot accurately reflect the breadth and depth of the lesion . AlthougThere were a large number of articles about endometrial diseases, but the level of the literature was uneven, and the description of ultrasonic characteristics was varied, without a unified consensus. It not only confused clinicians in interpreting ultrasound reports but also did harm to the development of these fields. Based on the above situation, the experts of the International Endometrial Tumor Analysis group (IETA) have written three normative descriptions of endometrial lesions and consensus literatures related to histopathology from 2010 to 2020 , 10, 13.In the past decades, there were several studies –23 on thSerum tumor biomarkers have been widely used in clinic, but there is no serum tumor biomarker with high specificity for endometrial cancer , 24, 25.The carbohydrate antigen 15-3 (CA15-3), which is the important specific tumor biomarker for breast cancer , has alsCA19-9 is an oligosaccharide-related antigen, which is closely related to digestive tract tumors such as pancreatic cancer and gastric cancer, and gynecological tumors. The alpha-fetoprotein (AFP) is an acidic glycoprotein synthesized from yolk sac and liver cells in embryo and is currently the most sensitive marker for the diagnosis of primary liver cancer. The correlation between AFP and endometrial cancer is still at the exploratory stage. Some studies have shown that the expression of AFP in endometrial carcinomas or carcinosarcomas is not uncommon .The squamous cell carcinoma antigen (SCC-Ag) is a sensitive marker of cervical and vulvar squamous cell carcinoma . It is oThe relevant studies , 34, 40 In this study, we converted the normalized description of IETA ultrasound characteristics into a simple scoring method based on the existing IETA-related research results –23 and tThis is a retrospective study. A total of 594 patients with uterine cavity or endometrial lesions were enrolled who were admitted to The Seventh Affiliated Hospital of Sun Yat-sen University or Shenzhen Hospital of Southern Medical University from January 2017 to June 2020. The retrospective study has been approved by the Medical Ethics Committee of the two hospitals (KY-2020-030-01 and NYSZYYEC20200029), and the need for informed consent was waived.The inclusion criteria for this study were as follows:(1) All patients underwent transvaginal/transrectal ultrasonography within 1 month before surgery.(2) All cases were examined for seven tumor biomarkers within 1 month before surgery.(3) The human chorionic gonadotropin (HCG) test was performed to exclude pregnancy-related diseases.(4) There was no adenomyosis in all cases; the abnormality of the endometrial–myometrial junction caused by adenomyosis was excluded.(5) The curettage or hysteroscopy or surgical excision was performed in enrolled patients, and the results were confirmed by pathological diagnosis or surgical records.Exclusion criteria were as follows:(1) All patients who did not meet the inclusion criteria(2) For medical ethical reasons, patients who were under legal age (<18 years old)(3) Patients who previously underwent abdominal surgical interventions, where the uterus has been removed(4) Patients who were allergic to ultrasound gel(5) Pregnant or lactating women or patients with a recent history of hormonal medication(6) Patients who had received radiotherapy, chemotherapy, or hormone therapy preoperatively or had tumors in other organs.We used three-dimensional ultrasound instruments . All instruments were equipped with high frequency intracavitary probe . All patients underwent transvaginal  ultrasound scan in a lithotomy position with an empty bladder, combined with transabdominal scan if necessary.Every assessment of the uterus should start with identification of the bladder and the cervix. The position of the uterus was noted and measurements taken. The uterus was scanned in the sagittal plane from cornu to cornu and in the (oblique) transverse plane from the cervix to the fundus. Having established an overview of the whole uterus, the image was magnified to contain only the uterine corpus. The magnification should be as large as possible, focusing on the area of interest . The proIn this study, we classified and scored the normalized description of IETA ultrasonic characteristics, according to IETA expert consensus literature , 10, 13,The research coordinator encoded the stored images to mask the personal information. Two senior sonologists who both had more than 10 years working experience in gynecology ultrasound analyzed all the ultrasound images independently without knowing other information after being fully familiar with the specific contents of IETA expert consensus on uterine cavity or endometrial lesions. The two observers scored the ultrasound images characteristics one by one according to the simple scoring diagnostic criteria, and the total score of lesions in each patient was finally calculated. The recorded results of the two observers were summarized. If there was any inconsistent result, the two observers discussed and negotiated, and the negotiated results were analyzed statistically.The results of serum CA125, CA15-3, CA19-9, HE4, SCC-Ag, AFP, and CEA were included.Detection Method: Five milliliters of venous blood of the patients was collected as a test sample and centrifuged at 3,000 rpm for 15 min. The serum was separated, and serums CA125, CA15-3, CA19-9, HE4, SCC-Ag, AFP, and CEA were detected by chemiluminescence. The testing instrument was Abbott I-2000 chemiluminescence instrument, and the reagents were as received.Criteria: Tumor biomarkers were positive when they exceeded the normal value.Reference values: CA l25 ≤ 35 U/ml; CA l5-3 ≤ 35 U/ml; CA l9-9 ≤ 35 U/ml; HE4: premenopause < 70 pmol/L, post-menopause < 140 pmol/L; SCC-Ag ≤ 1.5 ng/ml; AFP ≤ 20 ng/ml; CEA ≤ 5 ng/ml.In all cases of intrauterine adhesions, the results were confirmed by surgical records as no pathological specimens were available. The results of other lesions were confirmed by pathological results. Histopathological diagnoses of the specimens were obtained and served as reference standards. All diagnoses were made by specialized gynecological pathologists with more than 10 years’ experience, who were blinded to the results of the ultrasound examination. See All statistical analyses were performed with SPSS version 23.0 software . The quantitative data were expressed as mean ± standard deviation. The normal distribution and homogeneity of variance were tested. The independent-samples T-test or nonparametric test was used for the quantitative data. The values of categorical variables were presented by percentage (%); the chi-square test or Fisher’s exact test was used for data comparison.p < 0.05 was considered statistically significant.The ROC curve and the parallel test were used to analyze the sensitivity, specificity, and coincidence rate of simple ultrasound scoring method and simple ultrasound scoring method plus tumor biomarkers in the diagnosis of benign and malignant endometrial lesions; meanwhile, the respective area under the curve (AUC) was obtained. The ROC curve evaluated the diagnostic effectiveness of the value of AUC, which was between 1.0 and 0.5. When the AUC > 0.5, the closer AUC was to 1, the better the diagnostic effect. The AUC > 0.9 was of high diagnostic value. If the AUC was between 0.7 and 0.9, the diagnostic value was moderate. If the AUC was between 0.5 and 0.7, the diagnostic value was low. When AUC = 0.5, it indicated that the diagnostic method was completely ineffective and had no diagnostic value. There were 594 cases enrolled in this study; 475 cases were benign lesions, and 119 cases were malignant lesions. The endometrial atypical hyperplasia was classified as malignant lesions in this study because the current clinical treatment was based on malignant lesions. See p < 0.05). The demographic and clinical variables are shown in The differences between the benign and malignant groups were statistically significant in age, the proportion of patients before and after menopause, body mass index (BMI), the proportion of patients with irregular vaginal bleeding, and the number of deliveries   were benign. The blood flow in malignant lesions (color score: 2.84 ± 0.808) was significantly higher than that in benign lesions (1.72 ± 0.494).The vascular pattern of patients with endometrioid adenocarcinoma or other endometrial malignant lesions was dominated by multiple vessels  vascular pattern, accounting for about 94.4% (84/89). The endometrial vessels in patients with endometrial atypical hyperplasia were varied, 27.8% (5/18) were multiple vessels pattern, 33.3% (6/18) were single vessel pattern, and 38.9% (7/18) were scattered vessels pattern. The endometrial hyperplasia  was mainly of scattered vessels pattern (62.8%), the endometrial polyps were mainly of single vessel pattern (73.7%), and the submucous myomas were mainly of circular vessels pattern (77.1%).In the IETA ultrasound characteristics simple ultrasound scoring method, the average score of benign lesions was 3.879 ± 1.279, and the average score of malignant lesions was 9.676 ± 4.491. With ≥6.5 points as the cutoff value for the judgment of malignant lesions, the sensitivity, specificity, and coincidence rates were 76.5%, 96.0%, and 92.1%, and the area under ROC curve (AUC) was 0.935, as shown in p ≤ 1.000). The difference between CA19-9 and HE4 in benign and malignant lesions was statistically significant .As shown in With the value of CA19-9 ≥13.96 U/ml as cutoff value, the sensitivity, specificity, and coincidence rate of the diagnosis of benign and malignant endometrial lesions were 54.8%, 74.7%, and 70.7% and AUC of 0.620. With the value of HE4 ≥39.075 pmol/L as truncation value, the sensitivity, specificity, coincidence rate, and AUC of the diagnosis of benign and malignant lesions were 77.4%, 67.9%, 69.8%, and 0.796, respectively.When CA19-9 was combined with HE4 in parallel, the diagnostic sensitivity, specificity, and AUC were increased to 89.8%, 90.4%, and 0.805, respectively. As shown in Early diagnosis of benign and malignant lesions in uterine cavity or endometrium is of great significance for early clinical treatment and improvement of prognosis , 38. TheIn this research, we classified and scored the normalized description of IETA ultrasonic characteristics, according to IETA expert consensus literature, previous IETA-related research literature, and the previous research experience of this project group. We found that this simple scoring method could not only rapidly assess the benign and malignant lesions in uterine cavity or endometrium but also had high diagnostic accuracy . Dueholm et al. , 18 had p < 0.01). In most benign lesions, the color score was ≤2 points, while, the color score was ≥3 points in most malignant lesions. The results of Alcazar et al. . Thirty-one cases with uniform endometrial echogenicity (including three-layer pattern) were all benign lesions. Seven of 160 endometria (4.4%) with a linear endometrial midline were malignant lesions, and 2.7% (5/183) cases with a single vessel without branching on unenhanced ultrasound were malignant lesions. Our results above are similar to the research conclusion of Van den Bosch et al.  that somIn this research, we included the endometrial atypical hyperplasia in the malignant lesion group, based on its clinical treatment protocol, was similar to malignant lesions. If it was removed from the malignant group refering to the study of Sladkevicius et al. , the diaIn the simple scoring method of this study, we weighted the score according to the degree of correlation between IETA ultrasound characteristics and benign and malignant lesions. Endometrial thickness, endometrial–myometrial junction, “bright edge” sign, intracavitary fluid, color score, and multiple vessels pattern, which were significantly related to benign and malignant lesions, would be given points with greater weight. The indicators that were not significantly associated with the differentiation of benign from malignant may be assigned a less weighted score or no score.Although the endometrial midline appearance between benign and malignant lesions was statistically significant, the irregular or not defined endometrial midline appearance was also accounted for the majority of benign lesions (51.2%), leading to great difficulties in the identification of benign and malignant lesions. Moreover, in this study, we found that if the score was assigned to the endometrial midline appearance, and ≥9.5 points were used as the cutoff value for the judgment of malignant lesions, the sensitivity, specificity, and area under ROC curve (AUC) were 70.6%, 97.0%, and 0.931; it not only increased the evaluation time but also slightly decreased the diagnostic efficacy. Therefore, in this research, the endometrial midline appearance was not assigned to the score.In this study, HE4 had the highest diagnostic efficiency; it had higher sensitivity (77.4%) and better diagnostic efficiency  than any other single tumor biomarker. The results were similar to those of Ge et al. , Li et aThe other tumor biomarkers  had poor diagnostic efficacy. The overall diagnostic efficacy of serum tumor biomarkers was not high; as Kozakiewicz , and the rate of misdiagnosis was 4.0% (19/475). Among the 28 malignant cases missed, 14 cases were endometrial atypical hyperplasia, and 14 cases were Stage IA endometrioid adenocarcinoma. The possible factors for missed diagnosis were the following: all the missed cases were endometrial atypical hyperplasia or early stage lesions, endometrial thickness was not significantly thickened, there was no obvious invasion of the endometrial–myometrial junction, the lesions’ blood flow was not much, and the vascular pattern was dominated by single vessel or scattered vessels pattern, so the total score was not high and could not be distinguished from benign lesions.However, among the missed cases, there were 10 cases with CA19-9 value exceeding the threshold (13.96 U/ml), 8 cases with HE4 value exceeding the threshold (39.075 pmol/L), and 7 cases with CA19-9 and HE4 value both exceeding the threshold. Therefore, the sensitivity of the IETA ultrasound simple scoring method combined tumor markers HE4 and CA19-9 could be significantly increased (up to 97.6%), and the rate of missed diagnosis can be reduced.Among the 19 cases misdiagnosed, there were 11 cases of submucous myomas, 2 cases of endometrial polyp, 3 cases of endometrial polypoid hyperplasia, 1 case of endometrial simple hyperplasia, and 2 case of endometrial complex hyperplasia. The cases of misdiagnosis were mainly submucous myomas, which were endowed with high score in the scoring method due to the interruption of the endometrial – myometrial junction, leading to false positive results. However, the echoes of submucous myomas were mostly hypoechoic, and the vascular pattern was mostly circular vessels pattern, which were significantly different from the echoes of endometrium malignant lesions (mainly hyperechoic or mixed echogenicity) and the vascular pattern of lesions (mainly multiple vessels pattern). Therefore, in practical work, the rate of misdiagnosis could be reduced by combining with other ultrasonic characteristic of the lesions while assigning scores.There are many advantages of transvaginal ultrasound, such as low cost, clear imaging, non-invasive, convenient, and repeatable operation. The cost of serum tumor biomarkers examination is also relatively low; the results are fast and reliable.The two tests are available in the vast majority of hospitals, including remote ones with poor medical equipments. Although the IETA ultrasound characteristics simple scoring method has better specificity and diagnostic efficiency, its sensitivity is still deficient. The diagnostic efficiency of tumor biomarkers examination for endometrial and uterine cavity lesions is not good. Combined with these two methods, the specificity decreased slightly, but the diagnostic sensitivity can be greatly improved. The prognosis was good for those who were diagnosed with early stage EC, with a 5-year survival rate higher than 90% –9, but mThere were several limitations in this research. First, this study was a retrospective study, Thus, there were some biases ; for example, in the IETA 2010 consensus literature, it was suggested that a sonographic examination should preferably be performed in the early proliferative phase (cycle days 4–6) before the menopause, but we cannot guarantee that all premenopausal patients would be screened at the preferable time. In addition, the patient’s medical history, especially the history of hormone administration, can only be provided through clinical history data or return call. If the patient’s memory is incomplete, its accuracy may be different from the real situation. Second, the number of malignant cases in this study was relatively small, and it is relatively difficult to study the stage and grade of endometrial malignant lesions and histopathological types. In future studies, multicenter large-scale prospective studies are needed to conduct in-depth studies on these aspects.In IETA ultrasound characteristics simple scoring method, with ≥6.5 points as the cutoff value, it could quickly and accurately assess the benign and malignant uterine cavity and endometrial lesions, with high diagnostic value. The diagnostic efficacy of seven tumor biomarkers was all moderate. Combining with these two methods, the comprehensive diagnosis could improve sensitivity and accuracy and reduce the risk of missed diagnosis. In low-income countries or areas with poor health systems, they are often unable to purchase expensive medical examination equipments, such as MRI or PET-CT, etc.; the medical equipments there are relatively backward. They are eager for a cheap, quick and effective way to help them with their daily diagnostic work. In our study, the transvaginal ultrasound examination and tumor biomarker examination are relatively simple and inexpensive examinations with high comprehensive diagnostic efficacy. We hope that our comprehensive diagnostic method can be recommend in low-income countries or areas with poor health systems.The original contributions presented in the study are included in the article. Further inquiries can be directed to the corresponding authors.This study has been approved by the Medical Ethics Committee of the Seventh Affiliated Hospital of Sun Yat-sen University and Shenzhen Hospital of Southern Medical University (KY-2020-030-01 and NYSZYYEC20200029). Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.DL: study concepts and design, literature research, data collection, statistical analysis, manuscript writing. LZ: design work, data collection, statistical analysis. YZ: data collection and statistical analysis. YHu: data collection. KY: the research coordinator. WL: data analysis, data collection; SL: data collection, data analysis and manuscript editing; XG: statistical analysis and manuscript editing. YHa: project development, literature research, data management, and manuscript editing. All authors contributed to the article and approved the submitted version.This study was supported by the general programs from the Natural Science Foundation of Guangdong Province (2021A1515011585); Shenzhen Science and Technology Innovation Committee (grant nos. JCYJ20190814110207603 and JCYJ20190814111801681); the National Natural Science Foundation of China (grant no. 81972423); the clinical research start-up plan of Southern Medical University (Grant no. LC2016YM018); the incubation programme of National Natural Science Foundation of China from Shenzhen Hospital of Southern Medical University (grant no. PY2020ZY02); the social public welfare project of science and Technology from Shenzhen Baoan District (grant no. 2016CX301); the Grant of Shenzhen Key Laboratory of Viral Oncology (ZDSYS201707311140430); and the Grant of Shenzhen Sanming Medical Project (SM201702). The funding bodies had no influence on the study design or the collection, analysis or interpretation of data.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.The reviewer HX declared a shared affiliation, with several of the authors DL, YZ, YH, KY, and WL, to the handling editor at the time of the review.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.