To assess predictive value of clinical and pathological characteristics for metastatic radioactive iodine-refractory differentiated thyroid carcinoma (RAIR-DTC) in early stage retrospectively.We studied 199 metastatic DTC patients who were divided into two groups (TgAb negative and TgAb positive). The stimulated Tg (Sti-Tg) at the first and second radioiodine therapy (RIT) were defined as Sti-Tg1 and Sti-Tg2, the suppressed Tg (Sup-Tg) were designated as Sup-Tg1 and Sup-Tg2, while the TgAb were defined as TgAb1 and TgAb2, respectively. Univariate analysis and Logistic regression were used to investigate the effects of 13 observed factors to predict RAIR-DTC.In TgAb negative group, ROC curve analysis showed that cut-off values of age, Sti-Tg2/Sti-Tg1 and Sup-Tg2/Sup-Tg1 to predict RAIR-DTC were 40 years old, 57.0% and 81.0%, respectively. Age, extrathyroid invasion, Sti-Tg2/Sti-Tg1, Sup-Tg2/Sup-Tg1 and BRAF gene mutation were proved to be independent factors predicting RAIR-DTC. In TgAb-positive group, ROC curve analysis showed that cut-off values of age, TgAb1 and TgAb2/TgAb1 to predict RAIR-DTC were 55 years old, 297 IU/ml (14.8 times higher than the upper limit) and 53.6%, respectively.For TgAb-negative DTC, age over 40, extraglandular invasion, mutated BRAF gene, Sti-Tg decreased less than 43%, and Sup-Tg decreased less than 19% after the first two courses of RIT were independent predictors for RAIR-DTC. For TgAb-positive DTC, age over 55, extraglandular invasion, mutated BRAF gene, distant metastasis before RIT, TgAb level 14.8 times higher than the upper limit, TgAb dropped less than 46.4% after two courses of RIT were influencing factors. For decades, “thyroidectomy + radioiodine therapy (RIT) + L-Tcumented , 2. Howecumented , 4, and Each patient was provided with written information about the principles of the therapy and regulations of radiological protection. Further, written informed consent was obtained from all patients for participating the study. The selection and drop outs of study participants were shown in Flowchart Figure Figure 1This retrospective study was conducted between January 2003 and October 2019. There were 199 of 206 cases with complete follow-up data. Loss to follow-up rate was 3.4%. Collectively, 199 m-DTC patients  were enrolled. The average follow-up time was (50.2 ± 20.3) months. The inclusion criteria were as follows: (1) received total or near total thyroidectomy, (2) metastatic lesions existed, (3) received at least two courses of RIT, (4) both Tg and thyroglobulin antibody (TgAb) measured at the same time and scored according to the results. Patients were divided into TgAb negative group and TgAb positive group.131I-WBS showed focal or diffuse lung, lymph node, bone or other metastatic uptake, with or without other positive findings  and with or without Tg (TgAb) level elevation, (3) m-DTC patients diagnosed with different metastases site by post-therapeutic or diagnostic 131I-WBS after thyroid remnant ablation.Patients were defined as lung, lymph node, bone or other site of metastasis with the following facts : (1) metThe clinical and histopathological data evaluated in this study included gender, age, primary tumor size, extrathyroid invasion, single or multiple primary tumors, AJCC TNM staging , BRAF geA serum thyroid stimulating hormone (TSH) level of higher than 30 μIU/mL was achieved by thyroid hormone withdrawal (THW) before RIT. All patients were instructed with a low iodine diet from the beginning of THW till 3 weeks after RIT. Patients were administrated with a radioactive iodine dose of 3.7–7.4GBq (100-200mCi) according to the Chinese guideline . The secThe functional detection sensitivities of Tg and TgAb by electrochemiluminescence immunoassay  were 0.2 ng/mL and 20 IU/mL. TSH was determined by chemiluminescence immunoassay , with a measuring range from 0.35 to 4.94 μIU/mL. A serum stimulated or suppressed Tg, TSH, TgAb were usually followed 1 day before RIT and 2–3 months after RIT.131I-WBS was obtained by using Discovery NM/CT 670  equipped with high-energy parallel-hole collimators, and a 20% energy window was centered at 364 keV, at a table speed of 14 cm/min for a total time of 20 min (256×1024 matrix). Post-therapeutic whole-body scan (Rx-WBS) was performed 3-6 days after RIT (The patients were divided into TgAb negative group and TgAb positive group. The stimulated Tg (defined as Sti-Tg) at the first and second RIT were defined as Sti-Tg1 and Sti-Tg2, the suppressed Tg (defined as Sup-Tg) were designated as Sup-Tg1 and Sup-Tg2, while TgAb were defined as TgAb1 and TgAb2, respectively.131I contamination), (3) RAI was avidly concentrated in some lesions but not in others, and (4) metastatic disease progressed despite avidity of RAI.Till the last time of follow-up, RAIR-DTC was defined if the patient met any of the followings criteria : (1) theUnivariate analysis, t test and Mann-Whitney test were used for qualitative variables, and chi-square test was used to compare categorical data. Logistic regression was used for multivariate analysis. ROC (Receiver operating characteristic) curve analysis was used to determine the critical value for predicting RAIR-DTC. Statistical analysis was carried out using SPSS . A p value of less than 0.05 was considered significant.A total of 199 m-DTC patients  were involved in this study, 150 were in the TgAb negative group and 49 in the TgAb positive group. Among them, 4 cases were follicular thyroid carcinoma (FTC) and 195 cases were papillary thyroid carcinoma (PTC). A total of 77 patients were considered as RAIR-DTC. The average follow-up time was (50.2 ± 20.3) months. The clinical and pathological characteristics of the included patients were shown in Univariate analyses on the factors to predict RAIR-DTC were given in The results of TgAb positive group showed that cases with older age, extrathyroidal invasion, higher AJCC M stage, higher TgAb1 level, BRAF V600E mutation positivity, and lower TgAb2/TgAb1 would more likely progress to RARI-DTC . However, we found no statistically significant differences in gender, primary tumor size, primary tumor multifocality, AJCC T stage, AJCC N stage, lung metastasis size, and lymph node metastasis size. In specific, cases over 55 years old, with extraglandular invasion, positive BRAF gene mutation, distant metastasis before the first RIT, TgAb level 14.8 times higher than the upper limit, TgAb dropping less than 46.4% after the first two courses of RIT were influencing factors for becoming RAIR DTC.Data with statistical significance in the above univariate analyses were further assessed by ROC curve.In TgAb negative group, ROC curve showed that age, Sti-Tg2/Sti-Tg1 and Sup-Tg2/Sup-Tg1 had good performances in predicting RARI-DTC Figure 2In TgAb positive group, ROC curves were drawn regarding age, TgAb1 level and TgAb2/TgAb1 in predicting RARI-DTC Figure 3In TgAb negative group, multivariate logistic regression Table 4 Due to small number of cases in TgAb positive group, the logistic regression analysis was not conducted.131I therapy evaluation, under which circumstance the patients may have been exposed to unnecessary RAI and missed the opportunity to receive more effective interventions (such as targeted drug therapy). There is a need to be able to predict RAIR-DTC before 131I therapy and direct to a more individualized treatment  pathway (Regarding BRAFV600E mutation in DTC, literature indicates conflicting results. Some viewpoints suggest that BRAFV600E mutation is associated with DTC recurrence and poor prognosis –30. The  pathway , 32. The pathway , 34. OurIn our research, we also focused on whether extrathyroid invasion could lead to RAIR-DTC. Ha’s study suggested that aggressive histopathologic subtypes were significantly associated with the recurrent or metastatic lesions in RAI-negative DTC . RobertsLi et al.  evaluateThis study had limitations. Firstly, the design of retrospective studies has shortcomings and should be verified by future prospective studies. Secondly, the number of TgAb positive cases was small, and it was difficult to conduct multivariate analysis. Thirdly, there may be interactions between positive results obtained by univariate analysis.In summary, this study showed for TgAb-negative DTC patients, age over 40, extraglandular invasion, mutated BRAF gene, Sti-Tg decreased less than 43%, and Sup-Tg decreased less than 19% after the first two courses of RIT were independent predictors for RAIR-DTC. For TgAb-positive DTC patients, age over 55, extraglandular invasion, mutated BRAF gene, distant metastasis before the first RIT, level 14.8 times higher than the upper limit, TgAb dropped less than 46.4% after the first two courses of RIT were influencing factors.The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding authors.The studies involving human participants were reviewed and approved by Ethical Committee of Tianjin Medical University General Hospital. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.JC and RW contributed to the conception and design of the study. JC, RZ assisted with data acquisition. JC, RZ, and RW conducted the statistical analyses and drafted the manuscript. JT, ZM, QJ and GZ critically revised the manuscript. All authors read and approved the final manuscript and agree to be accountable for all aspects of the research in ensuring that the accuracy or integrity of any part of the work are appropriately investigated and resolved.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.


TaINVs were collinear with their ancestors in the synteny analysis, and we speculated that polyploidy events were the main force in the expansion of the TaINV gene family. Compared with TaCI, TaCWI and TaVI are more similar in gene structure and protein properties. Transcriptome sequencing analysis showed that TaINVs expressed in multiple tissues with different expression levels. Among 19 tissue-specific expressed TaINVs, 12 TaINVs showed grain-specific expression pattern and might play an important role in wheat grain development. In addition, qRT-PCR results further confirmed that TaCWI50 and TaVI27 show different expression in grain weight NILs. Our results demonstrated that the high expression of TaCWI50 and TaVI27 may be associated with a larger TGW phenotype. This work provides the foundations for understanding the grain development mechanism.Invertase (INV) irreversibly catalyzes the conversion of sucrose into glucose and fructose, playing important role in plant development and stress tolerance. However, the functions of INV genes in wheat have been less studied. In this study, a total of 126 TaINV genes were identified using a genome-wide search method, which could be classified into five classes  based on phylogenetic relationship. A total of 101  Sucrose is synthesized in the source leaves, loaded into the phloem for long-distance translocation, and unloaded into non-photosynthetic sink tissues such as roots, stems, flowers, fruits, and seeds . Many stArabidopsis thaliana [Oryza sativa [Zea may [Glycine max [Saccharum officinarum [Pisum sativum [Solanum tuberosum [Malus domestica [Populus [The invertase genes have been identified by genome-wide analyses in many plant species, including thaliana , Oryza sa sativa , Zea may[Zea may , Glycinecine max , Saccharicinarum , Pisum s sativum , Solanumuberosum , Malus domestica , and Pop[Populus . The inv[Populus . Ac-INVs[Populus . Moreove[Populus . Accordi[Populus . Meanwhi[Populus . The CIsCYT-INV1 in arabidopsis and rice resulted in a shorter primary root [AtVI2 cause differences in root length between divergent genotypes [INCW2 in maize reduced maize grain weight by 70% [OsINV3 lead to a shorter panicle with lighter and smaller grains [CsVI2 was upregulated by drought and the overexpression of CsVI2 could enhanced drought tolerance in cucumber seedlings [At-A/N-InvC plays an important role in the maintenance of mitochondrial reactive oxygen species homeostasis and the regulation of germination and flowering [At-A/N-InvE mainly maintains the carbon balance between plastids and cytoplasm [AtVI2 may play an important role in regulating stomatal opening in Arabidopsis [The invertase genes perform various roles in plants ,7,8,17. ary root ,21. Simienotypes . As for t by 70% , and mutr grains . Conversr grains ,26. INVseedlings . Recentllowering . At-A/N-ytoplasm . AtVI2 mbidopsis . Knockdobidopsis . The genbidopsis .Triticum aestivum L.) (2n = 6x = 42) is one of the most important planted crops worldwide. As the third largest cereal crop, common wheat accounts for approximately 20% of the total dietary calories and proteins worldwide [TaCwi-A1, located on wheat chromosome 2A, could explain 4.8% of phenotypic variance for kernel weight [TaCWI-4A and TaCWI-5D) were confirmed to be associated with TGW (thousand grain weight), and compared with Hap-4A-C haplotype, Hap-4A-T haplotype was associated with larger TGW and fewer seeds [Ta-A/N-Inv1 gene negatively regulates the disease resistance to wheat stripe rust [Common wheat  has facilitated the identification of wheat gene families at the genome levels . AlthougT. aestivum (IWGSC RefSeq v2.1) were obtained from the IWGSC dataset . The genomic sequences, protein sequences, and annotation files of Triticum urartu (G1812), Triticum dicoccoides (WEWSeq_v.1.0), and Aegilops tauschii (v4.0) were downloaded from the Ensembl Plants Database ). The longest splice variant of these four species was selected as a representative for subsequent analysis. The invertase protein sequence of rice, maize, Aarabidopsis, and soybean were download from Ensemble Plants.The genomic sequences, protein sequences, and annotation files of https://pfam-legacy.xfam.org/, accessed on 25 November 2021) and were used for HMMER search with a threshold of e < 1 × e−10. Meanwhile, the protein sequences of 17 INVs in Arabidopsis and 18 INVs in rice were used as queries to search against the local protein database using e < 1 × e−5, querry coverage >70% and identity >50% as the threshold. Finally, all candidate invertase obtained from the above methods were identified by CD-search .To identify INV members in common wheat and its relatives, two methods were used, namely, HMMER search and blastP analysis. In detail, the hidden Markov model (HMM) seed files including Glyco_hydro_32N (PF0000251), Glyco_hydro_32C (PF08244), and Glyco_hydro_100 (PF12899) were downloaded from the Pfam website . The phylogenetic tree was constructed with MEGA X software using the Maximum-like method, with default parameters and the bootstrap test executed by 1000 replications [https://itol.embl.de/upload.cgi, accessed on 1 November 2022). At the same time, a phylogenetic tree of TaINV was also construct with the same method.To analyze evolutionary relationships of the INV gene family, a phylogenetic tree was constructed based on the alignment of the 346 putative full-length amino acid sequences of rice, maize, soybean, Arabidopsis, common wheat, and its ancestors  were analyzed using the MCScanX (Multiple Collinearity Scan toolkit: http://chibba.pgml.uga.edu/mcscan2/) with a threshold e < 1 × e−5 and 5 hits. A collinearity map was drawn by Dual Systeny Plotter software in TBtools [Collinearity analysis between wheat, rice, maize, Arabidopsis, soybean, and its relatives comprising  and synonymous (Ks) was calculated. The timing of duplication events formula T = Ks/2λ × 10−6 Mya was used to calculate divergence time (T) in millions of years (Mya), where λ = 6.5 × 10−9 [The physical location information of all 5 × 10−9 .https://web.expasy.org/cgi-bin/compute_pi/pi_tool, accessed on 14 December 2021) [http://meme-suite.org/tools/meme, version 5.5.0, accessed on 5 August 2022). The repeat number was set to 0 or 1, the maximum number of motifs to 10, the motif width was 6 to 50, and the rest of the run parameters to system default.The molecular weight (MW), isoelectric point (pI), instability index (II), and grand average hydropathicity (GRAVY) were analyzed using the ExPASy online tools (er 2021) . At the http://www.softberry.com/, accessed on 14 December 2021). To further understand the function of N-terminal sequence, SignalP 6.0  was mainly used to predict the existence of signal peptide and TargetP-2.0  was used to predict the existence of mitochondrial transport peptide (mTP), chloroplast transport peptide (cTP), or cystoid coelomotor transport peptide (lTP). The presence of transmembrane helices (TMH) in proteins was predicted using TMHMM .The subcellular location of the TaINV proteins were predicted with the ProtComp 9.0 web tools (TaINV genes were obtained from gff3 files and displayed using TBtools. The 2 kb sequence upstream of the start codon is considered the promoter region and is extracted from the wheat genome (version 2.1). The cis-elements in the promoter sequences were identified in the PlantCARE database with the default parameters  [The exon/intron information of all er 2021) .TaINVs, multiple transcriptome data involved in different tissues  and of wheat cultivar Chinese Spring and responses to common stress , Blumeria graminis f. sp. Tritici (Bgt) and Puccinia striiformis f. sp. Tritici (Pst)) were downloaded from the Wheat Expression Browser  [TaINV genes were visualized by TBtools based on log2 (TPM + 1) values. For convenience, the heat map of genes in response to stress was drawn based on the log2 (TPM + 1) ratios of treatment to control groups.To understand the potential functions of er 2021) . The spaA pair of near isogenic lines (NILs), RHL81-L (TGW: 52.58 g) and RHL81-S (TGW: 33.35 g), produced by selfing of the RHL81, were used in expression analysis. Grains were marked at pollen mergence stage. Each sample contained 10 plants of uniform phenotype and the grains in the middle of spikelets shall be sampled at 4, 7, and 10 DPA (day post anthesis) and stored at −80 °C for RNA extraction. Total RNA was extracted using TRIZOL reagent and cDNA synthesis was performed with a reverse transcription reagent kit according to the manufacturers’ protocols.TaINV genes were analyzed by a real-time quantitative PCR (qRT-PCR). Specific primers of TaINV and TaActin gene  were designed with the Primer 5 program and shown in −ΔΔCT method. All data were analyzed and evaluated by GraphPad Prism . Two-tailed Student’s t test was performed to compare differences in gene expression.Nine TaCWI50 was amplified with the specific primers (TaCWI50-GFP-F/R) and inserted into pYJGFP vector using the ClonExpress II One Step Cloning Kit . The 35S::TaCWI50-GFP recombinant plasmid was used for substant experiments after sequenced. The recombinant plasmid 35S::TaCWI50-GFP and the control vector 35S::GFP were transformed into Nicotiana benthamiana leaf epidermal cells using Agrobacterium tumefaciens (GV3101) according to a previously described method [The full-length coding sequence (CDS) of T. aestivum, T. urartu, T. dicoccoides, and Ae. tauschii, respectively. To identify the INV genes in common wheat and its ancestors, four HMM seed files were obtained: Glyco_hydro_32C (PF08244), Glyco_hydro_32N (PF00251), Domain of unknown function (PF11867), and Glyco_hydro_100 (PF12899) by entering the INV protein sequence of Arabidopsis and rice. Then, 375 protein sequences were obtained by HMMsearch and BlastP. After removing the sequences with incomplete domain determined by Pfam and CDD databases, 126 TaINVs, 35 TuINVs, 65 TdINVs, and 32 AetINVs were identified and used for subsequent analysis   . The phyauschii) . The fivauschii) .TaVI1, TaVI2, and TaVI3 genes evolved earlier than other TaVI genes  and two dicotyledons    . Wheat a G. max) B. While ctively) C. Comparoccoides D. In addoccoides . The numon pairs .TaINV duplications genes varied from 0 to 0.8509, indicating that the duplicated TaINV gene has undergone purification selection  is a tyrosine-based lysosomal sorting signal and specifically exists at the N terminal of all TaVIs. The existence of P/V in WECP/VD in other species distinguishes CWI and VI, which becomes more complex in wheat. There is P or L in TaCWI and V or I in TaVI in wheat EC motif (motif 6). Motif 7 has four conserved hydrophobic amino acid  and two consecutive conserved hydrophilic amino acid (RR), which may have an important role in the correct folding of proteins. Motif 2, known as β-fructosidase motif, is found lacking in TaCWI10, TaCWI13, TaCWI21, TaVI29, and TaVI39. Motif 6 (EC motif) contained a catalytic site E that lacked in TaCWI58. Thus, we proposed that the enzyme catalytic activity encoded by these six genes is reduced or lost .In TaCI class, all genes contain the 10 motifs, with little difference in some amino acids. Aside from TaCI1 that lacks motif 10 and TaCI7 that lacks motif 1, the remaining 19 TaCIs contain all 10 motifs. Notably, six conserved amino acid residues  from five conserved motifs were regular varied within the CI gene family . These sTaINV genes exhibited different temporal–spatial expression patterns . For example, TaVI20, TaCWI33, TaCWI12, TaCI21, TaCI14, and TaCWI41 were specifically upregulated for drought, heat, cold, Pst, Bgt, and Fhb stress, respectively  exhibited grain-specific expression  are specifically expressed in grains, we speculate that elopment .TaCWI50, a signal peptide-absented INV, was expressed more highly in large grain lines than in small grain lines, we studied its subcellular localization. The results showed that TaCWI50 was located on the cell membrane. This is inconsistent with the software prediction results  are typical secretory proteins due to containing a typical signal peptide. However, some differences between CWI protein sequences of the invertase subfamily are obviously. Since  results  and indiTaINVs distributions among different chromosomes are imbalanced. The chromosome 4A with 16 TaINV genes has the highest number of any chromosome. Aside for TaCWI10, which is located in 4AS, the other 15 genes are located on chromosome 4AL. Among them, TaCWI32 collinears with TaCWI37 and TaCWI39, showing the collinear relationship between 4AL/4BS/4DS. Four TaCWIs (TaCWI33 to TaCWI35) were collinear with genes located in 5BL and 5D. As for TaVIs, except for TaVI6, TaVI10, and TaVI12, the remaining six genes collinear with genes in 7AS and 7DS. The collinearity between chromosomes of different homologous group may be due to the reciprocal translocations during wheat information [The INV genes were widely distributed in plant species, such as Arabidopsis , rice 66, maize ormation .TaINVs and 23 TuINVs, 67 TdINVs, and 30 AetINVs. The 106 TaINVs includ all TaCIs, 36 TaVIs, and 49 TaCWIs. We speculated that the other 20 TaINVs were generated by fragmented replication and tandem repeat replication according to the intraspecies collinearity analysis of wheat. In detail, 10 TaINVs including TaCWI7, TaCWI12, TaCWI17, TaCWI52, TaCWI58, TaVI12, TaVI21, TaVI23, TaVI25, and TaVI29 were generated by the tandem repeat of TaCWI6, TaCWI13, TaCWI16, TaCWI51, TaCWI57, TaVI11, TaVI20, TaVI22, TaVI24, and TaVI28, respectively. TaCWI10, TaCWI44, and TaVI29 were generated by the fragmented replication of TaCWI19, TaCWI33, and TaVI7 genes, respectively . Thus, we deduced that TaCWI4 and TaCWI22 are important for supplying a carbon source to developing filial tissues by cleaving unloaded sucrose in the apoplast. Further, TaCWI14, the orthologous genes of OsCIN2 gene, may also be a potential domestication gene and that such a domestication-selected gene can be used for further crop improvement. Here, the tissue expression profile analyses of the TaINV genes showed that many TaINV genes were highly expressed in spike and grain, suggesting that these genes may play important roles in regulating early grain filling and grain size. Although the detailed functions need to be tested in wheat, this provided a cue for dissecting the grain yield.The known functions of gene family members can be used to predict the functions of other genes on the same branch. Collinearity and phylogenetic tree analysis showed that  in rice , while Od weight . Here, wTaVI7/TaVI8 and TaVI28/TaVI29 gene pairs are not expressed in all tissues, while TaVI9/TaVI10 and TaVI26/TaVI27 are specific expressed in grains. Additionally, the four gene pairs do not respond to any stress. The other gene pairs had different expression patterns. Four genes including TaCWI13, TaCWI26, TaCWI52, and TaCWI58 show no functionalization in four gene pairs . The expression levels of two genes in tandem duplication gene pairs usually exhibited expression discrepancy in animal and model plants, indicating that the retention of gene duplicates might be associated to processes of tissue expression divergence ,55. In tTaVI21 was highly expressed in 15 different tissues, but the expression level of TaVI20 was low. Similarly, TaVI21 was strongly downregulated under cold stress, and TaVI20 did not response to cold stress. For plant development, TaCWI47 and TaCWI48 were differentially expressed with opposite expression patterns at DPA4 in RHL81L/S NILs, while TaCWI49 had anther-specific expression [TaCWI50 had grain-specific expression. This information gave us a cue that tandem duplication of TaINVs may be not only function in increasing levels of tissue specificity but also in genomic complexity, and therefore in increasing the adaptation of plants.However, the expression levels of 13 pairs of tandem duplication genes exhibited expression discrepancy. For instance, pression  and TaCWTaCWI50 and TaVI27 could be candidate genes relating to the grain-size trait. Although much work needs to be done to verify the detailed function in the future, this study here set an important base to understanding TaINV genes in the regulation of growth, development, and tolerance of various stresses.In summary, we proposed that TaINV genes and categorized them into five classes. The number of INV genes has increased dramatically in wheat, and we speculate that polyploidy events are the main force behind its expansion. Compared with CI, TaCWI and TaVI are more similar in gene structure and protein properties. TaINVs have different spatiotemporal expression patterns and play an important role in responding to biotic and abiotic stresses. Our results demonstrated that TaCWI50 was located on the cell membrane and its high expression may be associated with a larger TGW phenotype.In this study, we comprehensively characterized 126


Ovarian cancer is a highly lethal female malignancy with high rates of advanced stage and recurrent disease. Ovarian cancer is characterized as poorly responsive to immunotherapy. This is hypothesized to be secondary to its highly immunosuppressive and phenotypically “cold” tumor microenvironment (TME). Tumor-associated macrophages (TAMs) are an integral component of the ovarian cancer TME, and predominantly display an M2 protumor phenotype. Ovarian cancer cells and TAMs interact via a complex network of signaling cytokines. Ovarian cancer TAMs are linked to tumor metastasis, angiogenesis, chemoresistance, and poor prognosis. Due to their profound role, TAMs represent a therapeutic target for ovarian cancer immunotherapy. Strategies for targeting TAMs include increasing phagocytosis, decreasing recruitment, decreasing macrophage survival, and repolarizing TAMs to an antitumor phenotype.The tumor microenvironment (TME) has been implicated to play an important role in the progression of ovarian cancer. One of the most important components of the TME is tumor associated macrophages (TAMs). Phenotypically, macrophages are broadly categorized as M1 pro-inflammatory or M2 anti-inflammatory, based on the cytokines and chemokines that they secrete. The tumor microenvironment is associated with macrophages of an M2 phenotype which suppress the surrounding immune environment, assist tumor cells in evading immune targeting, and support tumor growth and metastasis. Contrarily, M1 macrophages help mount an immune response against tumors, and are associated with a more favorable prognosis in solid tumors. One of the characteristic indicators of a poor prognosis in ovarian cancer is the overrepresentation of M2-type TAMs. As such, therapeutic modalities targeting TME and TAMs are of increasing interest. Pharmacological approaches to eliminate TAMs, include decreasing macrophage survival and recruitment and increasing phagocytosis, have been underwhelming. Clinical strategies targeting these macrophage subtypes via repolarization to an M1 antitumoral state deserve increasing attention, and may serve as a new modality for immunotherapy. Ovarian cancer is the deadliest gynecological malignancy in the US, with an estimated 19,880 new cases and 12,810 deaths in 2022 ,4. Due tMacrophages encompass a significant component of the human innate immune system, and are an attractive target for new immunotherapies . The priMacrophages that either reside or are recruited to tumor tissue are described as tumor-associated macrophages (TAMs), and can display a wide functional phenotypic expression depending upon the microenvironment . TAMs apThe tumor immunologic microenvironment has been recognized as an increasingly important aspect of cancer biology, and as a potential target for precision therapy. TAMs are significant components of tumor immunology, and despite the traditional role of macrophages as anti-tumoral and cytotoxic, TAMS have demonstrated considerable protumoral properties ,15. TAMsThe traditional framework for understanding macrophages, and thus TAMs, is the breakdown into M1 and M2 macrophage populations. Macrophages exhibit high plasticity, and can be “activated” or “polarized” to their phenotypic profile, both in vivo and in vitro, via various cytokines, such as IFNγ, lipopolysaccharide (LPS), and others, for M1 and IL-4, IL-10, IL-13 ,19,23,24Historically, the discovery and categorization of M1 versus M2 was related to their metabolism of arginine . M1 macrBesides arginine metabolism, there are several metabolic pathways and metabolites that influence macrophage polarization . M1 and CD4 T helper cells produce a large subset of immune system cytokines that can be categorized by their downstream effects. Th1 cytokines  produce a proinflammatory state, whereas Th2 cytokines  result in an anti-inflammatory response . An uncoPrior studies have indicated that high infiltration of TAMs within tumors is associated with adverse clinical outcomes  Figure . AdditioA retrospective study analyzing patients with stage III-IV ovarian cancer utilized CD68 and CD163 as M1 and M2 macrophage markers, respectively, to analyze the expression of TAMs within tumor samples. When comparing high vs. low CD163 (M2 TAMs), there was a significant difference in both progression-free and overall survival, both significantly higher in the low CD163 groups . MultivaTAMs originate either from resident tissue macrophages or circulating bone-marrow-derived monocyte precursors . Tissue-TAMs are the predominant immune system cell in the ovarian cancer TME. Data from The Cancer Immunome Atlas (TCIA) and compiled by authors at MD Anderson demonstrate that macrophages make up 39% of the immune cell type in ovarian cancer. This is compared to the second most—CD4 T cells, at 12% . Of these TAMs, the majority (51%) were M2, compared to 25, 20, and 4% for M0, M1, and monocytes, respectively . These dThe interaction between tumor cells and their associated tumor microenvironment is complex and mediated by a number of intracellular signaling pathways  10,22].,22.10,22In addition to exosomes, multiple secreted proteins have been found to be involved in cancer cell-mediated M2 polarization. Increased IL-4 signaling from ovarian ID8 cancer cells in mice induced increased PI3K pathway expression, inducing an M2 phenotype . Human eGATA3, an oncogenic protein, is highly expressed in high-grade serous ovarian cancer (HGSOC) patients. GATA3 is primarily secreted by TAMs, and has been linked to tumor growth, angiogenesis, and chemoresistance . TAMs reN-acetylaspartate, which acts synergistically with IL-10 to polarize macrophages to an M2 with high expression of glutamine synthetase [Metabolic crosstalk between TAMs and ovarian cancer also strongly influences the preponderance of M2 TAMs in the TME. Ovarian cancer cell secretion of hyaluronic acid increases membrane cholesterol efflux from macrophages. In turn, cholesterol upregulates IL-4 secretion and downregulates IFNγ activity via STAT6 and PI3K pathways . Glutaminthetase .The interaction between ovarian cancer cells and TAMs is driven by a complex cytokine network that is often mutually reinforcing. For example, ovarian cancer cells are capable of recruiting macrophages via CC-chemokine ligand 2 (CCL2)/Monocyte chemoattractant protein-1 (MCP-1), yet also polarize macrophages to an M2 state ,59,60. TTumor-associated macrophages contribute significantly to metastasis and disease progression ,61. In vM2 TAMs in coculture with SKOV3 ovarian cancer cells increased STAT3 activation. STAT3 is a critical point of convergence for a number of oncogenic pathways . TAMs alIL-10 is an anti-inflammatory cytokine that directly inhibits the cytotoxic activity of natural killer cells and macrophages . IL-10, TAMs also upregulate TGF-β production via STAT3 upregulation. TGF-β inhibits cytotoxic activity of NK and lymphocytes . TGF-β, TAMs play a pivotal role in the transcoelomic  spread of ovarian cancer by supporting cell survival and invasiveness ,16. TAMsTAMs are found in malignant ascitic fluid, and support further ovarian cancer cell migration by releasing tumor growth factor—beta-induced (TGFβI) and tenascin C . TGFβI iIn summary, TAMs are instrumental in ovarian cancer disease progression. TAMs function as a powerful driver of immunosuppression within the TME. TAMs also enhance metastasis via multiple signaling pathways, which is highly pertinent in ovarian cancer, a disease often discovered only after metastatic spread. TAMs play an important role in chemoresistance in ovarian cancer ,21,61. CUbiquitin protein ligase E3 component n-recognin 5 (UBR5) is an E3 ligase that plays a role in platinum resistance in ovarian cancer . AdditioIn vitro, apoptotic ovarian cancer cells induce macrophages to polarize to the M2 phenotype. Troublingly, this suggests that apoptotic cells induced by chemotherapy may contribute to the enhanced formation of M2 macrophages, and a further immunosuppressive TME . DifferePlatinum-based chemotherapy remains a cornerstone of ovarian cancer treatment, with the development of platinum resistance as a dominant prognostic marker . EvidencImmunotherapies have successfully treated a variety of diseases, including certain cancers, for decades . Ovarian−2 cisplatin and 600 mg m−2 cyclophosphamide in the upfront adjuvant setting demonstrated an improvement in 3 year PFS . Toxicity was deemed acceptable, though noted a near-ubiquitous flu-like syndrome in the experimental arm [One historical approach to overcome the immunosuppressive TME was the systemic administration of immune-activating cytokines. IFNγ polarizes macrophages to an M1 state, and prior studies demonstrated early success with this approach, including administration of intraperitoneal IFNγ at second-look laparotomy . Anotherntal arm . Howeverntal arm . A finalntal arm .Precision therapeutics that target TAMs are a proposed strategy at overcoming the cold immunophenotype TME. Early-phase clinical trials are underway exploring various macrophage-centric approaches, including targeting M2 macrophages directly via CSF-1R pathway, anti-CD47 antibodies to enhance macrophage phagocytosis, and anti-CCL2 antibodies to decrease macrophage recruitment  Figure . CC-chemThere are several pre-clinical and early clinical trials investigating the restoration of phagocytosis in TAMs via the inhibition of the CD47/SIRPa pathway ,115. CD4As mentioned previously, the CSF-1/CSF-1R axis is the primary regulator of macrophage recruitment and differentiation. Due to its cornerstone role in TAM development, there has been considerable interest in targeting the axis for cancer therapy ,121. PreOverall, the recent clinical evidence of TAM therapeutic strategies, such as CSF-1R inhibition and CD47 targeting, has been underwhelming . AdditioAn additional therapeutic framework with promise is a strategy for repolarizing protumor M2 macrophages to a tumoricidal M1 phenotype . A higheToll-like receptor (TLR) 7/8 agonists are immunostimulatory therapeutics with the capacity to repolarize macrophages to an M1 state. However, TLR 7/8 agonists have poor pharmacokinetic profiles and significant off-target effects. Large, anionic liposomes loaded with resiquimod, a TLR 7/8 agonist, were able to target and repolarize TAMs to an M1 state when administered intraperitoneally in murine ovarian cancer models . AdditioMultiple groups have taken an interest in the role of NF-kβ in the immune targeting of cancer cells.  The NF-kco-glycolic acid-based anionic nanoparticles have shown selective accumulation in ovarian tumor TAM mouse models [More targeted approaches to modulation have demonstrated promise. Bromodomain containing 4 (BRD4) protein, an epigenetic regulator, has been associated with M2 to M1 polarization . BRD4 ine models . Encapsue models .Clodronate is a bisphosphate with current clinical use in the treatment of osteoporosis, Paget’s disease, hypercalcemia, multiple myeloma, and breast cancer . BisphosImmune-cell-derived exosomes have been proposed as TAM repolarization therapeutics . ExosomeOvarian cancer is a highly lethal female malignancy that is unresponsive to current immunotherapy approaches. A major reason for this cold immunophenotype is the highly immunosuppressive TME, which, in turn, is dominated by TAMs. TAMs have been directly linked with increased tumor invasiveness, enhanced metastasis, angiogenesis, chemoresistance, and worse clinical survival outcomes. Early clinical TAM-specific therapeutic strategies, including increasing macrophage phagocytosis and restricting recruitment, have been underwhelming. However, macrophage phenotype modulation, in which protumor M2 TAMs are repolarized to an antitumor M1 state, is a promising strategy, and warrants further investigation.


Aspergillus nidulans urea transporter, UreA. To deepen the structural characterization of UreA, we employed the artificial intelligence (AI) based AlphaFold2 (AF2) program. In this analysis, the resulting AF2 models lacked inward- and outward-facing cavities, suggesting a structural intermediate state of UreA. Moreover, the orientation of the W82, W84, N279, and T282 side chains showed a large variability, which in the case of W82 and W84, may operate as a gating mechanism in the ligand pathway. To test this hypothesis non-conservative and conservative substitutions of these amino acids were introduced, and binding and transport assessed for urea and its toxic analogue 2-thiourea, as well as binding of the structural analogue acetamide. As a result, residues W82, W84, N279, and T282 were implicated in substrate identification, selection, and translocation. Using molecular docking with Autodock Vina with flexible side chains, we corroborated the AF2 theoretical intermediate model, showing a remarkable correlation between docking scores and experimental affinities determined in wild-type and UreA mutants. The combination of AI-based modeling with classical docking, validated by comprehensive mutational analysis at the binding region, would suggest an unforeseen option to determine structural level details on a challenging family of proteins.Through a combination of comparative modeling, site-directed and classical random mutagenesis approaches, we previously identified critical residues for binding, recognition, and translocation of urea, and its inhibition by 2-thiourea and acetamide in the  Bacteria, fungi and plants are able to use urea as the sole nitrogen source for the biosynthesis of nucleic acids, lipids and proteins. This substance occurs in nature as a product of animal, plant and microbial metabolism. Additionally, it accounts for more than 70% of the applied synthetic nitrogen fertilizer ,2. Howev+ symporters within the sodium:solute symporter (SSS) superfamily -urea (55.0 mCi mmol−1) was purchased from Moravek Biochemicals, Brea, CA, USA. [14C]-urea uptake assays were performed in A. nidulans germinating conidiospores. Initial velocities were measured after 2 min of incubation at 37 °C with a [14C]-urea concentration of 1.0 μΜ, and are expressed as % of the wt value, in pmoles min−1/107 conidiospores. Apparent mK/i values were obtained directly by performing and analyzing [14C]-urea uptakes , from IC50 values in the presence of various concentrations (0.5–3000 μM) of non-labelled substrates, as previously described    and in tw/v) polyacrylamide gel) and electroblotted onto a nitrocellulose membrane , which was then blocked with 5% non-fat dry milk. Immunodetection was performed with primary mouse monoclonal antibodies against anti-GFP (Roche Molecular Systems) or against anti-tubulin  and a secondary sheep anti-mouse IgG HRP-linked antibody . Blots were developed with the Amersham ECL Western Blotting detection kit and images were acquired with the GBox Chemi XT4 System and analyzed with the GeneSys software .Total protein extracts were obtained from 200 mg of grinded mycelia, as described in Apostolaki et al., 2012  and prothttps://micro-manager.org/, Madison, WI, USA).Samples for epifluorescence microscopy were incubated in glass-bottom dishes in liquid minimal media with proline (5 mM) as nitrogen source and appropriate supplements at 25 °C for 14–16 h and protected from light. Observations were done in an Olympus inverted microscope CKX31, which belongs to the Advanced Bioimaging Unit, Institut Pasteur de Montevideo, and is equipped with a U-MNIBA3 filter for GFP and a Hamamatsu Orca Er camera, and the Micro-Manager software  (https:/


Recent studies have reported that hyperglycemia-induced cellular stress stimulates pyroptosis, and different signaling pathways have been shown to play crucial roles in regulating pyroptosis. This review summarized and discussed the molecular mechanisms, regulation, and cellular effects of pyroptosis in diabetic microvascular complications, such as diabetic nephropathy, diabetic retinopathy, and diabetic cardiomyopathy. In addition, this review aimed to provide new insights into identifying better treatments for diabetic microvascular complications.Pyroptosis is an inflammatory form of programmed cell death that is dependent on inflammatory caspases, leading to the cleavage of gasdermin D (GSDMD) and increased secretion of interleukin (IL)-1 Diabetes is a low-grade inflammatory disease that seriously affects the quality of life of 463 million adults aged 20–79 years . Diabetiβ, IL-18, and lactate dehydrogenase [Pyroptosis is a type of programmed cell death and its morphological characteristics are mainly caused by cell membrane rupture, chromatin condensation, nuclear fragmentation, and substantial release of cellular contents, including interleukin (IL)-1rogenase . Pyroptorogenase . Recent rogenase , rheumatrogenase , and nerrogenase , are assrogenase . This reShigella flexneri [tZychlinsky et al. first discovered the phenomenon of pyroptosis in 1992, which was initially considered apoptosis in macrophages infected by flexneri . Studiesflexneri . In 2015flexneri , 12. GSDflexneri . The GSDflexneri .Inflammation is a complex process that reflects local and systemic responses to different immunological and nonimmunological stimuli. Pyroptosis, which is essentially an inflammatory reaction, has provided a new perspective for research on the pathophysiological mechanisms of DMCs. Pyroptosis mechanisms include canonical and noncanonical pathways. The difference between the two pathways is primarily due to their triggering factors. The two pathways are independently discussed below, and the details can be found in Figures β and pro-IL-18 are sheared to activate IL-1β and IL-18, respectively, by activating caspase-1, and GSDMD is cleaved into GSDMD-N to form pores on the cell membrane [The canonical pyroptosis pathway is caspase-1-dependent and is triggered by inflammasomes . In thisβ from macrophages was inhibited in Casp11−/− C57BL/6 or Casp1−/− Casp11129mt/129mt mice infected with Gram-negative bacteria and that cell death induced by caspase-11 activation was independent of NLRP3 and ASC [The noncanonical pyroptosis pathway is caspase-11- or caspase-4/caspase-5-dependent (caspase-11 in mice and caspase-4/5 in humans) . In 2011 and ASC . Lipid A and ASC . Notably and ASC .Recently, although limited, studies have attempted to explore the relationship between pyroptosis and DMCs and their potential mechanisms. Studies on pyroptosis and its associated diseases are summarized in this review.β2 microglobulin. In addition, some biomarkers related to the pathogenesis of DN, such as kidney injury molecule 1 (Kim-1), neutrophil gelatinase-associated lipocalin (NGAL), tissue inhibitor of metalloproteinases-2, insulin-like growth factor-binding protein 7 (IGFBP-7), vascular endothelial growth factor (VEGF), transforming growth factor-β (TGF-β), monocyte chemoattractant protein-1(MCP-1), and inflammatory cytokines, such as tumor necrosis factor (TNF)-α, MCP-1, and ILs , have also attracted significant attention [DN is one of the most prevalent and serious microvascular complications associated with diabetes. Its early pathological characteristics include basement membrane thickening, increased mesangial matrix production, and extracellular matrix accumulation, with the subsequent development of glomerulosclerosis and tubulointerstitial fibrosis, eventually leading to proteinuria and irreversible renal damage. The early clinical diagnosis of DN is based on canonical biochemical markers, such as glomerular filtration rate, urinary microalbumin, urinary microalbumin to urinary creatinine ratio, serum creatinine, urinary cystatin C, and serum ttention , 25. Howin vitro study has reported that LPS directly induces pyroptosis in vascular endothelial cells [Glomerular endothelial cells (GECs) are located within the glomerulus and are the first layer to be involved in glomerular filtration. Recent findings suggest that both cell loss and inflammation in GECs are important causes of DN , and thaal cells . Becauseal cells . Anotheral cells .β in podocytes derived from patients with DN, db/db mice, and streptozotocin (STZ)-induced mice/rats [β in podocytes, whereas inhibition or knockdown [κB) signaling and NLRP3 inflammasome to alleviate kidney injury and podocyte pyroptosis in DN [κB pathway in podocytes in DN [κB pathway [Abelmoschus manihot (TFA) alleviate podocyte pyroptosis and injury by adjusting methyltransferase-like protein 3 (METTL3)-dependent m6A modification and regulating NLRP3-inflammasome activation and phosphatase and tensin homolog (PTEN)/phosphoinositide 3-kinase/protein kinase B (Akt) signaling [Podocytes are the outer glomerular filtration barriers. Podocyte fusion and foot process effacement cause proteinuria, and cell death and inflammation are the underlying mechanisms . Podocytice/rats , 32. Higice/rats , D-ribosice/rats , and visice/rats  levels dnockdown  of NLRP3nockdown , and casnockdown  improvesnockdown . Additionockdown . Thus, tnockdown . There inockdown . Forkheais in DN . Moreoveis in DN . A recenis in DN . Furtheres in DN . Atorvases in DN . Catalpo pathway . The totignaling . As podoβ, and pro-IL-1β, in GMCs [β activation and decreases renal fibrosis [Glomerular mesangial cells (GMCs) play a pivotal role in maintaining the basic structure of the glomerulus. Increasing the mesangial matrix and GMCs proliferation promotes DN development. Inflammation is an important factor in GMC proliferation . Previou in GMCs , 48, whefibrosis . Nuclearfibrosis . Furtherfibrosis , 51.β increases significantly in both STZ-induced DN rats and high glucose-treated RTECs [in vivo and in vitro [κB signaling pathway also modulates GSDMD-mediated pyroptosis in RTECs [Renal tubular epithelial cells (RTECs) are more vulnerable to death because they are inevitably stimulated by various pathogenic factors, such as toxins, hypoxia, and metabolic disorders. In patients with diabetes, the expression of IL-18, an inflammatory cytokine released during pyroptosis in renal tubular cells, increases significantly (approximately 83%) , suggested RTECs . Inhibitin vitro , 55. Morin vitro . The TLRin RTECs . Recent in RTECs , 58. Morin RTECs . Specifiin RTECs  or hirudin RTECs . In summDR is a hallmark complication of diabetes and a leading cause of vision loss in adults. Loss of retinal pericytes is one of the earliest changes associated with DR, and it has been postulated to initiate or trigger microaneurysm formation, abnormal leakage, edema, and ischemia, provoking proliferative neovascularization in the retina. Although the pathophysiological mechanisms of DR are complex, vascular endothelial damage, increased vascular permeability, and neovascularization are the most common phenomena.α, IL1β, IL-6, IL-8, chemokines, C-C motif ligand-2, intercellular cell adhesion molecule-1, and vascular cell adhesion molecule-1 [Hemoglobin A1c (HbA1c) is the only validated systemic biomarker for DR progression. However, only 6.6% of the variation in the risk of DR is explained by HbA1c levels . In addilecule-1 . Furtherlecule-1 , 63. Howβ increases in patients with DR [β, improve retinal layer thickness, and ameliorate blood-retinal barrier permeability [Hyperglycemia-induced cell pyroptosis in the retina has been well demonstrated . The den with DR . A simil with DR . Further with DR  or sileneability , thus efeability . Moreoveeability . Furthereability . Furthereability . In addieability , 72.Although inhibition of caspase-1 or NLRP3 has been suggested to delay DR progression, a series of issues remain to be solved. First, current studies have mainly focused on the NLRP3-caspase-1 signaling pathway in DR. Therefore, the role of the noncanonical pyroptosis pathway in this disease remains unknown. Moreover, as inflammasome activation does not always trigger pyroptosis, other signaling pathways involved in pyroptosis should be investigated. Second, the regulatory mechanism of pyroptosis is currently focused on noncoding RNA, and other regulatory mechanisms are yet to be defined. Third, GSDMD is the ultimate executor protein in pyroptosis, and its function in DR requires further in-depth study. In addition, although studies have indicated the influence of resolvin D1, minocycline, caspase 1, and NLRP3 inhibitors (such as Mcc950), or H3 relaxin  on blockDC is the main cardiovascular complication that occurs in approximately 60% of patients with well-controlled diabetes, resulting in systolic and diastolic dysfunctions, which are independent risk factors for any vascular disease or hypertension . Increasβ, activin A, ROS-induced inflammatory cytokines , galectin-3, suppression of tumorigenicity 2 (sST2), lncRNAs, and microRNAs [A broad spectrum of cardiovascular biomarkers that have been described in patients with DC includes brain natriuretic peptide (BNP), cardiac troponins , and matrix metalloproteinases (MMPs), particularly MMP-9. Some biomarkers related to the pathogenesis of DR have been reported, such as cardiotrophin-1, IGFBP7, TGF-icroRNAs . HowevericroRNAs . We hopeβ increased significantly in cardiomyocytes of STZ-treated diabetic rats and that NLRP3 silencing or inhibition of caspase-1 reduced their expression, alleviated left ventricular dysfunction, and ultimately reversed myocardial remodeling in DC. Similar findings were observed in a DC model developed from C57BL/6J mice [β1/Smads pathway in cardiac fibroblasts is repressed by KCNQ1OT1 knockdown [Due to the undefined pathophysiology of DC, several studies have attempted to investigate the role of pyroptosis in DC. In early 2014, Luo et al.  reported/6J mice , 80. Apa/6J mice . The stu/6J mice . Similar/6J mice . Moreovenockdown . Other nnockdown , miR-9 [nockdown , GAS5 [8nockdown , and MIAnockdown , are alsnockdown . In addinockdown .Many drugs have been demonstrated to have some effects against pyroptosis (e.g., metformin , exendinin vitro and in vivo. Based on an enhanced understanding of these existing results, we further explore how pyroptosis and inflammasome are activated, including some novel studies related to pyroptosis regulation and its relationship with DMCs, to provide a promising avenue for DMC prevention and treatment. Three new insights have been discovered regarding the regulation of pyroptosis in DMCs:The important causes of DMCs are oxidative stress and inflammation resulting in pyroptosis. Based on the antioxidative stress and antiinflammatory strategies, we determined effective drugs or small-molecule compounds against pyroptosis to improve DMCs and further explored the regulation of pyroptosis and its potential mechanisms, which will open up a new direction for the prevention and treatment of DMCs.The pro-inflammatory effect of pyroptosis is a key factor in the development of DMCs. Some studies have found that several upstream regulatory signaling proteins, noncoding RNAs, and circRNAs play a role in the upregulation of pyroptosis. Furthermore, autophagy plays a protective role in the inhibition of NLPR3 overactivation . Therefoβ and IL-18. IL-1β and IL-18 are two essential pro-inflammatory cytokines that are upregulated in tissue-resident cells of patients and animals with diabetes and are further induced by positive feedback to pyroptosis to form inflammatory storms [The inflammatory response is an important factor in the development of DMCs. Pyroptosis maintains or expands inflammation by tightly controlling the inflammatory response via the release of IL-1y storms . Based oIn conclusion, the pathophysiological mechanisms underlying DMCs are complex and unclear. However, pyroptosis, a type of programmed cell death that triggers inflammatory reactions, provides a novel perspective for determining the mechanisms and therapeutic targets of DMCs . These eNevertheless, whether some promising pyroptosis biomarkers can be used as novel targets for diagnosing and treating DMCs requires further investigation, particularly in animal experiments and clinical studies. Further investigations into the exact molecular and regulatory mechanisms of pyroptosis are necessary for DMCs. Furthermore, GSDMD, as the ultimate pyroptosis protein, should be investigated for its function in DMCs and to determine whether inhibiting GSDMD is a more valuable therapeutic target for these complications. Moreover, inhibition or activation of the pyroptosis pathway affects various aspects of physiology and can have different effects. It is necessary to consider the overall body balance when studying drugs that target the pyroptosis pathway in DMCs.


NT5C3B, CNP, ACLY, and GNB1L maintained overexpression at the mRNA level in the cell lines and in patients with ALL. It is noteworthy that SALL2 showed very high expression in T-ALL, while JUP was highly expressed in B-ALL lineages. Interestingly, the latter correlated with worse survival in patients. This provided evidence that the measurement of these genes has high potential for clinical utility; however, their expressions should first be evaluated with a sensitive test in a more significant number of patients.Acute lymphoblastic leukemia (ALL) in children or adults is characterized by structural and numeric aberrations in chromosomes; these anomalies strongly correlate with prognosis and clinical outcome. Therefore, this work aimed to identify the genes present in chromosomal gain regions found more frequently in patients with acute lymphoblastic leukemia (ALL) and ALL-derived cell lines using comparative genomic hybridization (CGH). In addition, validation of the genes found in these regions was performed utilizing RNAseq from JURKAT, CEM, and SUP-B15 cell lines, as well as expression microarrays derived from a MILE study. Chromosomes with common gain zones that were maintained in six or more samples were 14, 17, and 22, in which a total of 22 genes were identified. From them,  Acute lymphoblastic leukemia (ALL) results from the malignant transformation and proliferation of hematopoietic stem cells (HSCs) in the bone marrow, blood, and extramedullary sites, and it is characterized by genetic mutations . It was Although there may be predisposing factors to develop the disease, such as the presence of some syndromes  [ETV6-RUNX1) [TCF3-PBX1) [IGH-IL3) [MLL); the intrachromosomal amplification of chromosome 21 (iAMP21) [CDKN2A/B [Some typical anomalies useful in a clinical setting for B-ALL, in addition to hyperdiploidy and hypodiploidy, are the translocations t (CR-ABL1) , t , t1;19)F3-PBX1) , and t(5IGH-IL3) ; the rea(iAMP21) ; and theF3-PBX1) MLL) or the deletion of CDKN2A/B [TAL1), (11p13) (LMO2), (10q24) (TLX1/HOX11), (5q35) (TLX3/HOX11L2) [NUP214-ABL1) [NOTCH1) [FBXW7) and (10q23) (PTEN) [Although T-ALL shares abnormalities with B-ALL, such as the rearrangement of tv;11q23)  , fusion 14-ABL1) , mutatio (CDKN2A/MO2), 10q , which is characterized by allowing, with a high resolution, the identification of small numerical aberrations in a genome . CGH canTherefore, the objective of this study is to identify the common chromosomal gain regions most frequently found in bone-marrow-derived samples from patients with ALL and the leukemia-derived cell lines JURKAT and CEM using CGH arrays; to validate expressions at the transcriptome level of the genes included in the chromosomal gains using RNAseq and microarray expression analysis; and to correlate the gene expressions with overall survival.The present study used bone marrow samples from ALL patients without prior treatment that were isolated by density gradient centrifugation with Ficoll-Paque™ PLUS  and cryopreserved in liquid nitrogen from a previous study by the working group; the age, gender, leukemia classification, immune phenotype, and blast percentage of each patient included in this study are visualized in 2 culture flasks  in RPMI 1640 (Cat.11875-093) supplemented with 10% inactivated fetal bovine serum (FBS) and 100 U/mL penicillin/1 mg/mL streptomycin  at 37 °C in a 5% CO2 atmosphere. The cell lines used in this study are commercially available at the ATCC and were kindly donated by Prof. Dr. Henning Walczak . Authentication was performed by Multiplexion GmbH (https://www.multiplexion.de accessed on 17 October 2022).Cell lines derived from T-ALL (JURKAT and CEM) and B-ALL (SUP-B15) were cultured in 25 cmTM12  machine, and scanning was executed using an MS 200 NimbleGen Microarray Scanner with a resolution of 2 µm (Roche Applied Science).Genomic DNA was obtained using a Quick-gDNA™ MiniPrep kit  according to the manufacturer’s instructions. DNA was stored at 4 ºC until use. Sample preparation and hybridization were performed as described in detail in the NimbleGen Arrays User Guide . Briefly, 1 µg DNA from each leukemia-derived sample was taken and labeled with Cy3 (cyanine 3), and the reference DNA provided by the kit was labeled with Cy5 (cyanine 5). Subsequently, DNA probes were hybridized on a NimbleGen HG18 WG CGH Array . After 72 h of hybridization, slides were washed and spin-dried in a SlideWasherThe values of the Log2 ratios of the probes (Cy3/Cy5), signal intensities, and chromosomal gains and losses were calculated and visualized using DEVA software, version 1.2.1 (Roche Applied Sciences).In brief, local polynomial regression fitting (LOESS) spatial normalization was performed, followed by Qspline normalization of the intensities of all microarrays. After ratio calculation of the test and reference samples, all data from all containers were merged into a single container. Next, a segment tree was built using SegMNT, with 500 as a maximum number of segments, a minimum number of differences between segments of 0.2 (Log2), a minimum number of probes in segments of 5, a permutation number of 10, and stringency for the percentile of 0.9.https://genome.ucsc.edu/ accessed on 1 September 2022) with the GRch/38hg 38 genome version as reference. The raw and processed data obtained in this study were already deposited in the Gene Expression Omnibus (GEO) NCBI database repository under accession number GSE185274. To increase the certainty of our findings, we included samples derived from the bone marrow of adult patients with ALL without prior treatment (available under accession number GSE75671) [Finally, the genes present in the gain regions were identified utilizing the UCSC Genome Browser database (SE75671) . The samSE75671) .https://cran.r-project.org/web/packages/circlize/index.html accessed on 17 October 2022), RColorBrewer (https://cran.r-project.org/web/packages/RColorBrewer/index.html accessed on 17 October 2022), GenomicRanges (https://bioconductor.org/packages/release/bioc/html/GenomicRanges.html accessed on 17 October 2022), data.table (https://cran.r-project.org/web/packages/data.table/index.html accessed on 17 October 2022), RLumShiny (https://cran.r-project.org/web/packages/RLumShiny/index.html accessed on 17 October 2022), and grDevices (https://uribo.github.io/rpkgshowcase/graphics/grDevices.html accessed on 17 October 2022). To discriminate between a region with or without gain, we use a cutoff point of 1.5.The circular multitrack plots shown in were generated using genome-wide DNA copy numbers normalized to Log2 utilizing R, version 4.1.3, and RStudio software (2021.09.0). The libraries utilized were Circlize (https://www.ncbi.nlm.nih.gov/gds with public access on 31 October 2021) and identified with accession number GSE189641 [RNA sequencing of JURKAT and CEM cell lines was performed using the NovaSeq 6000 Illumina platform . These sequences were deposited in the GEO NCBI repository . First, the FastQC tool  was used to determine the quality of the sequences [A bioinformatic analysis was carried out as follows: raw reads were analyzed using the Galaxy Europa open-source platform  and RStudio software (2021.09.0) utilizing Rsubread library   using FPJUP and the reference genes (RPLP0 and RPS18) are represented in Total RNA was isolated from peripherical mononuclear cells derived from individuals without leukemia and from JURKAT, CEM, and SUP-B15 cell lines with a Quick-RNA mini prep plus kit . Afterward, cDNA was obtained with a Transcriptor First Strand cDNA Synthesis Kit . qPCR assays were performed with a LightCycler 2.0 (Roche Diagnostics) instrument using a LightCycler FastStart DNA Master plus SYBR Green I kit . The sequences of the primers used to amplify http://r2.amc.nl accessed on 1 September 2022). This database is derived from a microarray design (Affymetrix HG-133 Plus 2.0) that includes samples from adults with ALL without treatment. Expressions of the 22 genes identified in the common gain regions were evaluated by comparing controls (n = 71) with B-ALL (n = 427) or T-ALL (n = 165) samples. A one-way ANOVA was applied, and the expressions were transformed into Log2.To analyze the expression of each gene in patients with ALL, we used the open-access NCBI GEO database with access number GSE13159 ,45 and Rwww.bloodspot.eu accessed on 17 October 2022. The files used for the analysis were GSE13159 for the leukemia lineages (MILE project) [Hierarchical trees for each gene were constructed using BloodSpot, an online database of gene expression profiles and transcriptional programs for healthy and malignant hematopoiesis  availablproject)  and GSE2project) .http://www.r-project.org accessed on 17 October 2022), by utilizing the GEO NCBI database with access number GSE34861 [p < 0.05 were taken as statistically significant.The association between overall survival and gene expression was calculated using the Kaplan Scan tool, available in Statistical Software Environment R, version 2.4.1 (Microarrays of CGHs were performed in two ALL-derived cell lines (JURKAT and CEM) and in 10 samples derived from the bone marrow of ALL patients to determine chromosome gains. As described in DEVA v 1.2 of the common gain zones in each sample are visualized in Once it was determined that chromosomes 14, 17, and 22 contained the most frequent gains, we continued to determine each patient’s gain regions. After the regions were determined, we identified regions that overlapped in at least six samples, a condition fulfilled only for chromosomes 14, 17, and 22. To improve the certainty of our results, we analyzed data from the GSE75671 study, which contained CGH arrays of bone marrow from adult patients with ALL and was performed with the same platform and Genome CGH arrays used in our study. From the 18 samples analyzed (nine T-ALL and nine B-ALL), eight and seven out of nine presented gain regions in T-ALL and B-ALL patients, respectively, for chromosome 14. Regarding chromosome 17, we identified nine (T-ALL) and five (B-ALL) samples and, in chromosome 22, four and five, respectively , and the following six genes were recognized for chromosome 14: P3H4, NT5C3B, CNP, ACLY, RTL10, COMT, and GNB1L genes maintained overexpression in the three cell lines, regardless of subtype. Interestingly, JUP mainly had a high expression in SUP-B15, while SALL2 mainly had a high expression in JURKAT and CEM. For data validation, JUP expression was further determined by qPCR; as shown in JUP expression was very high in SUP-B15 cells (changes of 46.9-fold taking RPS18 as reference gene and 28.7-fold taking RPLP0), while in CEM and JURKAT, it was almost undetectable.Derived from the RNAseq analysis, heatmaps were created from JURKAT (T-ALL), CEM (T-ALL), and SUP-B-15 (B-ALL) cell lines versus nonleukemia controls . As observed in METTL3, NT5C3B, CNP, JUP, KLHL10, KLHL11, and GNB1L), while SALL2 and ACLY were overexpressed only in T-ALL patients. The gene that was found to be most significantly overexpressed in B-ALL was JUP. Unexpectedly, TOX4, DNAJC7, TTC25/ODAD4, TXNRD2, and TANGO2 were underexpressed in ALL patients. No significant differences were found in other genes.The relative expression at the RNA level of each gene was determined using accessible microarray expression databases from the MILE project, as described in Materials and Methods. As observed in SALL2 and JUP since, during normal hematopoiesis, both genes were highly expressed only in hematopoietic stem cells (HSCs)  and ALL hyperdiploid samples. However, JUP was principally found to be highly expressed in ALL t, ALL hyperdiploid, and ALL t, followed by Pro- and Pre-B-derived samples. In addition, moderate expressions were observed in some AML subtypes, as seen in Once we identified the genes with statistically significant high expressions in B-ALL or T-ALL patients, we were interested to know the expression of each gene during normal hematopoiesis and to compare it with the expressions in all the leukemia lineages. Each gene was analyzed utilizing BloodSpot, as detailed in Materials and Methods. After evaluation, we determined that evident differences were observed just in SALL2 and JUP expressions in leukemia patients in contrast to healthy bone marrow samples, we determined whether the expressions of these genes were related to better overall survival. Since survival studies require a long follow-up period and we do not yet possess these data in our working group, we decided to search for a free database containing this information. After an exhaustive search, only one free database on the follow-up of adult B-ALL patients was found . After Kaplan–Meier curve analysis, as shown in JUP was statistically significantly associated with poorer overall survival.Since we saw relevant differences in the Acute lymphoblastic leukemia (ALL) involves the disruption of differentiation in a clonal lymphoid population in the early stages  that canRegarding the results visualized in NT5C3B, CNP, ACLY, JUP, KLHL11, RTL10, and GNB1L.In this study, it was of our interest not only to identify gains in genes, but also to validate their expressions at the mRNA level. As expected, we found genes that had already been widely reported, which validated our study. However, the originality of this work is that we identified genes that had not been previously linked to leukemia or any other cancer type, as seen in NT5C3B , which has until now only been involved in processes of the respiratory tract and atherosclerosis [KLHL11  [There is limited information about two of the genes mentioned above: clerosis ,59, and mber 11) , part ofmber 11) . To dateCNP, the enzyme 2′,3′-cyclic nucleotide 3′ phosphodiesterase [CNP-positive patients had better survival rates than individuals with CNP-negative tumors [CNP expression correlated with better overall survival (data not shown).Regarding esterase , it has e tumors . AlthougACLY, which translates the enzyme ATP citrate lyase and is responsible for the synthesis of cytosolic acetyl-CoA [ACLY upregulation has been shown to promote metastasis and invasion and to inhibit apoptosis in prostate [Concerning etyl-CoA , an assoetyl-CoA . In otheprostate , colon [prostate , breast prostate , and esoprostate . Specifiprostate . In contJUP/Plakoglobin, a gene that produces the protein γ-catenin and is homologous to β-catenin [BCR-ABL1” genetic abnormality (through regulation of MYC and BIRC5/survivin) in B-ALL, being proposed as a potential therapeutic target [JUP expression in prostate cancer is controversial since changes depending on the stage [JUP was found to be highly expressed in the SUP-B15 cell line and B-ALL individuals . This gene translates to a BH3 protein, a motif that is part of the proapoptotic proteins Bad, Bik, and PUMA. The role of the BH3 protein in ALL, individually or as a motif of proapoptotic proteins, has not been described; nevertheless, PUMA levels were elevated in chronic lymphoblastic leukemia (CLL) [Another gene that we found to have a high expression was ia (CLL) . These dGNB1L (G protein beta 1 subunit) in the cell lines and ALL patients; however, there are no reports in the literature that have studied the expression of this gene in any type of cancer. Most studies on GNB1L are related to psychiatric disorders reported, for example, in schizophrenia [In our study, we found overexpression of ophrenia  and autiophrenia . Only onophrenia ; thus, wSALL2 , which is a member of the spalt/sal family of transcription factors associated with cell differentiation, development, and stemness [SALL2 with TCR-α (TRA-SALL2) was one of the most recurrent fusions reported in this pathology [SALL2 have been found in esophageal cancer [SALL2 was overexpressed in T-ALL-derived cells and because, during normal hematopoiesis, its expression is limited to HSCs, we believe that the expression of this gene could be useful as a lineage marker and as a potential therapeutic target.Concerning stemness , its expathology . In addil cancer , breast l cancer , testicul cancer , and glil cancer . In contl cancer , as welll cancer  and orall cancer . Since wCOMT and P3H4 , no association has been described with any type of leukemia; however, in lung and bladder cancer, the upregulation of mRNA and protein levels have been associated with the promotion of proliferation, migration, and invasiveness [P3H4 helped reduce cell invasion through miR-133a and miR-1a [On the other hand, there were two genes in which overexpression was determined in leukemia-derived cell lines but was not confirmed in patients: and P3H4 . With reukocytes ; in peditoxicity . In endotoxicity  and breatoxicity , some popoptosis . Regardisiveness ,95. On td miR-1a .METTL3 (N6-adenosine-methyltransferase) and KLHL10 (Kelch-like family member 10). The former methylates primary microRNAs (pri-miRNAs) that promote the initiation of miRNA biogenesis [METTL3 mRNA and protein expression was increased in acute myeloid leukemia (AML) cells compared to healthy hematopoietic stem and progenitor cells [ETV6/RUNX1-positive patients when compared to controls [KLHL10, there is limited information about its function, and there are no studies that associate it with cancer or any other pathology; to date, it has been related only to spermatogenesis and male infertility [In addition, we found two genes statistically significantly overexpressed in B- and T-ALL patients but not in the cell lines: ogenesis ; alteratogenesis , colorecogenesis , prostatogenesis , pancreaogenesis , ovarianogenesis , and esoogenesis . Furtherogenesis  and gastogenesis  have beeor cells , and a hcontrols . Based ocontrols ,109. Regertility ,111, andTOX4, DNAJC7, TTC25, TXNRD2, and TANGO2 , it is characterized by its participation in mucociliary clearance [TXNRD2/TRXR2), an important antioxidant enzyme that controls the levels of cellular reactive oxygen species (ROS), it was identified as being hypomethylated in CLL [TANGO2 (transport and organization homolog of the golgi complex 2), polymorphism in “p. Ser17Ter” was associated with an aggressive profile of prostate cancer [Unexpectedly, although our study observed gains in genomic DNA , lower ed TANGO2 A,B. Regaetection . In the learance . Intereslearance . Regardid in CLL . Furtherd in CLL , as welld in CLL . Concerne cancer . AccordiSALL2, NT5C3B, CNP, ACLY, JUP, KLHL11, RTL10, and GNB1L. Of these, it is worth mentioning that a hierarchical tree analysis showed very high expressions of SALL2 in T-ALL and JUP in B-ALL patients, as well as in HSC, but not in any other cell lineage generated during normal hematopoiesis. Moreover, it is essential to highlight that the overexpression of JUP was strongly related to poor overall survival in B-ALL patients. Deepening the study of these genes are relevant constitutes a fertile field of research to investigate how their alterations are related to this pathology and to evaluate with more sensitive tests their application in the diagnosis and prognosis of ALL, as well as their utility as therapeutic targets.This study identified genes present in common chromosomal gains detected through CGH microarrays in ALL patients and two classical leukemia-derived cell lines and validated them using expression microarrays and RNAseq. The expressing genes that we thought could be clinically relevant were


Rosa rugosa (RrOSC) provides a starting point for the triterpenoid pathway, which contributes to the medicinal value of the extraction of tissues of Rosa rugosa. However, the structure and function of key RrOSCs of active triterpenoids remain ambiguous. In this study, a total of 18 RrOSC members with conservative gene structures and motifs were identified based on the genome of Rosa rugosa. The RrOSCs were located on three chromosomes including two gene clusters that derived from gene replication. The phylogenetic relationship divided RrOSCs into six groups, and the RrOSCs of GI and GIV that were represented by lupeol or α-amyrin were identified as likely to include candidate genes for producing active triterpenoids. Considering the high expression or specific-tissue expression of the candidates, RrOSC1, RrOSC10, RrOSC12, and RrOSC18 were considered the key genes. RrOSC12 was identified in vitro as lupeol synthase. The results provided fundamental information and candidate genes for further illustration of the triterpenoid pathway involved in the pharmacological activities of Rosa rugosa.The oxidosqualene cyclase family of  Sterols and triterpenes are important components of plant metabolites, and nearly 200 different skeletons of these compounds have been identified . In addiOSC genes have been identified in plants and their catalytic function were mostly identified by heterologous expression in yeast. The Arabidopsis thaliana genome contains 13 OSC genes, including cycloartenol synthase (AtCAS1), lanosterol synthase (AtLSS1), and β-amyrin synthase (LUP4), and several OSCs with mixed functions  diviunctions ,10,11,12 product . Furtheroduction . Rosa rugosa is a shrub of Rosaceae and native to North China, Northeast China, Japan, Korea, and Russia, and has a long history of application as a medicinal plant. Its petals and roots have been widely used to staunch bleeding [R. rugosa root extract, the active triterpenoids Kaji ichigoside F1, rosamultin, euscaphic acid, and tormentic acid showed anti-injury and anti-inflammatory effects on animal models; among these compounds, rosamultin had a strong inhibitory effect on human immunodeficiency virus-1 protease [R. rugosa. However, the OSC genes of R. rugosa (RrOSCs) involved in the synthesis of pharmacological terpenoids  haven’t been reported. This study was aimed at discovering the potential key RrOSCs in the active terpenoids synthesis pathway of R. rugosa by systematic analysis of the RrOSC family.bleeding , reduce bleeding ,17. In tprotease ,19. ThesRrOSC genes were identified, and the predicted proteins ranged from 540 to 770 amino acids in length and from 62.283 to 85.712 kDa in molecular weight  were divided into 6 Groups (GI–GVI), which were named by common catalytic function based on the phylogenetic branches  or GVI (specific to monocotyledons).A total of 18 r weight . All OSCbranches . OSCs inRrOSCs was in the range of 14–21. The SQHop_cyclase_C and/or SQHop_cyclase_N domains located by constant exon number (18–20 exons) indicated gene structures of RrOSCs were conserved (RrOSCs composed of motifs 3-8-5-10-2 (corresponding to SQHop_cyclase_C) and motifs 6-7-4-1 (corresponding to SQHop_cyclase_N) were highly aligned. Interestingly, Group I members lack one motif5 in the N-terminal compared while other RrOSCs comprised of two motif5. And motif6 and/or motif7 were absent in SQHop_cyclase_N of Group I members RrOSC1 and RrOSC3.The exon number of onserved b. Analysonserved a showed RrOSCs are distributed on three  of all seven chromosomes  were not discussed further. RrOSC9, RrOSC10, and RrOSC13 are expressed in all tissues and all stages in a middle or high abundance (minimum TPM > 10), indicating their constitutive expression pattern.To detect the candidate branches . RrOSC2,RrOSC6, RrOSC10, and RrOSC13 in the lateral branches of R. rugosa showed two expression patterns. The expression of RrOSC6 and RrOSC13 increased from the first lateral branch to the third lateral branch. The expression of RrOSC10 was the highest, decreased from the primary lateral branch to the secondary lateral branch, and increased from the secondary lateral branch to the tertiary lateral branch. The three genes all showed the highest abundance in the third lateral branch, and only RrOSC6 specific expressed in branches.The highly-expressed RrOSC10, RrOSC12, RrOSC13, and RrOSC18 were high (FPKM > 50), and RrOSC12 was strongly expressed in TIR but almost not at all in OR. In flowers, the expression levels of RrOSC9, RrOSC10, and RrOSC13 were higher. The expression levels of RrOSC9 and RrOSC13 were high in the early stage, decreased at the end of the flowering stage, and reached a maximum at S2 and S3. The expression of RrOSC10 was the highest, and it increased significantly at the end of the flowering stage and reached the maximum at S7. In the lateral branches and petals, more than half of the genes were not expressed or had low expression.In roots  of R. rugosa was more than the 14 members found in Arabidopsis and 11 members in rice. A comparison of gene structure and motifs showed that the gene structure difference of RrOSCs in the same subgroup didn’t affect their highly-conserved domains including consistent motif structure. Additionally, many RrOSCs in the same subgroup shared more similar expression patterns, such as two different subgroups of GI: RrOSC7, RrOSC11, and RrOSC17 all highly expressed in flowers or RrOSC6, RrOSC10, and RrOSC18 all highly expressed in roots (or branches).The RrOSC1-RrOSC6, RrOSC10, and RrOSC18 were clustered with MdOSC3, EjAS, and MdOSC1 of Rosaceae plants as a subgroup. MdOSC1 yields α-amyrin, β-amyrin, and lupeol at a ratio of 85:13:2 [EjAS produces α-amyrin and β-amyrin at a ratio of 17:3, and MdOSC3 manufactures α-amyrin, β-amyrin, and lupeol at a ratio of 85:14:1 [RrOSC2-RrOSC3 and proximal duplication genes RrOSC6, and RrOSC10 indicated that gene duplications contributed to the functional redundancy of the seven α-amyrin synthase candidates. RrOSC7, RrOSC8, RrOSC11, and RrOSC17 combined with MdOSC4 and MdOSC5 were clustered into another subgroup. The main product of MdOSC4 is germanicol, that of MdOSC5 is lupeol, and both contain the by-product β-amyrin. Thus, the four candidates are unlikely to be α-amyrin synthases. According to the structural comparison, the rosamultin of R. rugosa should be formed from α-amyrin through cytochrome P450 oxidation to introduce functional groups and finally glycosylated by glycosyltransferase. Except for the four low abundance genes, above, RrOSCs, RrOSC1, RrOSC10, and RrOSC18 were considered as the candidate genes of the rosamultin synthesis pathway. The OSCs in the same subgroup in GI could produce mix-products with accordant main components.  85:14:1 . This suOSC of R. rugosa (RrOSC12) belongs to GIV, whose members would catalytic substrate to produce lupeol. The yeast expression proved its catalytic activity of lupeol. Though the lupeol synthase activity in vivo of RrOSC12 needs to be studied further, its remarkably high mRNA abundance in TR and the lupenone and betulinic acid (downstream products of lupeol) accumulation in TR (unpublished metabolome data) indicated that lupeol contributes partly to the medicinal value of R. rugosa roots. In addition, the chromosome terminal locations (near telomere) of RrOSC12 provide insight into DNA methylation, which could be the entry point for further exploring the pathway gene cluster to jointly modify the substrate .Lupeol is an important pentacyclic triterpenoid. The applications of lupeol derivatives in the field of medicine are extensively studied because of their pharmacological activities, including antinephrotoxicity, antihepatotoxicity, and anticancer, anti-inflammatory, anti-heart disease, antiarthritis, and antidiabetes properties . Lupeol OSCs is increasingly apparent, e.g., MdOSC1 [enzymes cycloartenol synthase gene of Panax ginseng and β-amyrin synthase genes of Glycyrrhiza glabra, and P. ginseng [R. rugosa that has been established by our own lab, we would like to overexpress RrOSC12 in R. rugosa hairy roots to increase lupeol production. In addition, the two α-amyrin synthase candidates RrOSC10/18, which were highly expressed in roots, should be tested further for increasing the potential of α-amyrin and that of the downstream product rosamultin.The enormous metabolic engineering potential of , MdOSC1 ,23, enzy ginseng ,25. BaseR. rugosa “Zi Zhi” were planted in germplasm in the resource nursery of Yangzhou University , Yangzhou, China. Samples including roots (OR), primary lateral branch (PB), secondary lateral branch (SB), tertiary lateral branch (TB), flowers of seven stages (S1–S7), and roots (TR) of tissue culture seedlings (two-month-old micro-cutting seedings) were collected and frozen using liquid nitrogen, and then stored at −80 °C for RNA extraction (The three-year-old cutting seedings of traction .RrOSCs were predicted by HMMER 3.0 from the proteome of R. rugosa “Zi Zhi” (unpublished) using the hidden Markov model of the squalene-hopene cyclase N-terminal domain (PF13249) and squalene-hopene cyclase C-terminal domain (PF13243) [http://pfam.xfam.org/, accessed on 1 September 2022) and the Conserved Domain Database [OSC genes were checked by manual inspection. The isoelectric point and molecular weight of RrOSC proteins were obtained by ProtParam tool of Extasy [PF13243) . The canDatabase ,28. The f Extasy .https://mafft.cbrc.jp/alignment/server/index.html, accessed on 1 September 2022) [OSC genes of Arabidopsis thaliana, Lotus japonicus, Panax ginseng, Abies magnifica, Adiantum capillus-veneris, Avena strigose, Betula platyphylla, Costus speciosus, Cucurbita pepo, Dioscorea zingiberensis, Glycyrrhiza glabra, Kalanchoe daigremontiana, Kandelia candel, Luffa cylindrica, Oryza sativa, Pisum sativum, Polypodiodes niponica, Rhizophora stylosa, Ricinus communis, Artemisia annua, Aster sedifolius, Bruguiera gymnorrhiza, Euphorbia tirucalli, Medicago truncatula, Nigella sativa, Polygala tenuifolia, Solanum lycopersicum, Vaccaria hispanica, Olea europaea, Taraxacum officinale, Aster tataricus, Stevia rebaudiana, Malus pumila, and Eriobotrya japonica are shown in RrOSC proteins and 78 OSC proteins of different plant species were aligned using the MAFFT algorithm (er 2022) . The phyer 2022) , with 10http://meme-suite.org/, accessed on 1 September 2022) with the default parameters and a maximum motif number of 8 [RrOSCs were illustrated by the gene structure view tool of TBtools toolkit 1.086 [RrOSCs  of the whole genome, and the syntenic regions were predicted by the MCScanX tool of TBtools toolkit 1.086 . The genR. rugosa “Zi Zhi” using HISAT 2.2.1 . The sequencing quality was examined by Q30 value, percentage of clean reads, and alignment coverage  were extracted by the MiniBEST Universal RNA Extraction Kit  for transcriptome libraries building and sequencing on the Illumina NovaSeq 6000 platform. The raw reads could be accessed in the SRA of NCBI (PRJNA725330) and BIG Sub of China National Center for Bioinformation (PRJCA012932). After low-quality reads filtering, clean reads were used for analysis. Based on clean reads, read counts of each gene were calculated by mapping to the genome of coverage . FragmenRrOSC12 was cloned by the polymerase chain reaction (PCR). The coding sequence of RrOSC12 was sub-cloned to pESC-leu and transformed to yeast strain AM94. Culture extracts of the RrOSC12 expressed yeast were derivatized with trimethylsilylating agents and submitted to gas chromatography–mass spectrometry analysis.