GPR37 is an orphan G protein-coupled receptor (GPCR) that is implicated in different physiological pathways in the brain, and has been linked to various malignancies. The aim of this study was to explore the relationship between GPR37 gene expression and the clinicopathological factors, patient prognosis, tumor-infiltrating immune cell signature GSEA and methylation levels in glioma.Background: Gliomas are the most frequently diagnosed primary brain tumors, and are associated with multiple molecular aberrations during their development and progression. Methods: We explored the diagnostic value, clinical relevance, and molecular function of GPR37 in glioma using TCGA, STRING, cBioPortal, Tumor Immunity Estimation Resource (TIMER) database and MethSurv databases. Besides, the "ssGSEA" algorithm was conducted to estimate immune cells infiltration abundance, with 'ggplot2' package visualizing the results. Immunohistochemical staining of clinical samples were used to verify the speculations of bioinformatics analysis.GPR37 expression was significantly higher in the glioma tissues compared to the normal brain tissues, and was linked to poor prognosis. Functional annotation of GPR37 showed enrichment of ether lipid metabolism, fat digestion and absorption, and histidine metabolism. In addition, GSEA showed that GPR37 was positively correlated to the positive regulation of macrophage derived foam cell differentiation, negative regulation of T cell receptor signaling pathway, neuroactive ligand receptor interaction, calcium signaling pathway, and negatively associated with immunoglobulin complex, immunoglobulin complex circulating, ribosome and spliceosome mediated by circulating immunoglobulin etc. TIMER2.0 and ssGSEA showed that GPR37 expression was significantly associated with the infiltration of T cells, CD8 T cell, eosinophils, macrophages, neutrophils, NK CD56dim cells, NK cells, plasmacytoid DCs (pDCs), T helper cells and T effector memory (Tem) cells. In addition, high GPR37 expression was positively correlated with increased infiltration of M2 macrophages, which in turn was associated with poor prognosis. Furthermore, GPR37 was positively correlated with various immune checkpoints (ICPs). Finally, hypomethylation of the GPR37 promoter was associated with its high expression levels and poor prognosis in glioma.Results: GPR37 had diagnostic and prognostic value in glioma. The possible biological mechanisms of GPR37 provide novel insights into the clinical diagnosis and treatment of glioma.Conclusion:  Accordin, grade  and 3; AGPR37 is a member of G-protein-coupled receptors (GPCRs), also known as Parkinson’s related endothelin like receptor (Pael-R). It is highly expressed in the brain and is related to neurological diseases such as Parkinson’s disease and autism [GPR37 in lung adenocarcinoma (LUAD) cells inhibited the malignant behavior [GPR37 in gastric cancer cells is linked to peritoneal metastases and poor prognosis [GPR37 as a potential prognostic biomarker for lung adenocarcinoma and human multiple myeloma [GPR37 in the U251 glioma cell line accelerated cell cycle progression, activate AKT pathway and promote proliferation [GPR37 in the genesis and progression of glioma.d autism . Knockinbehavior , whereas myeloma , 9. Up-rferation . HoweverGPR37 in glioma using bioinformatics.To that end, we investigated the prognostic significance and putative biological functions of http://gepia2.cancer-pku.cn/#index) was used to validate GPR37 mRNA expression levels in cancer and normal tissues [GPR37 between the glioma and normal tissues was analyzed by combining data from the GTEx (http://commonfund.nih.gov/GTEx) and The Cancer Genome Atlas (TCGA) , which had been uniformly processed by the Toil process in UCSC Xena (https://xenabrowser.net/datapages/) [Data from GEPIA2 ( tissues . The difapages/) –14. DataKaplan-Meier curves for the overall survival (OS) were plotted based on TCGA data, and compared using Cox regression. The R survival (version: 3.2-10) and survminer (version: 0.4.9) packages were used for statistical analysis and visualization.GPR37 the protein expression and distribution were analyzed in the HPA database (https://www.proteinatlas.org/) [Immunohistochemical data of as.org/) .Specimens of tumor and adjacent tissues were collected from 38 patients in the Second Affiliated Hospital of Guangzhou Medical University, who had undergone curative surgery from 2020 to 2022 in our hospital, which was approved by Institutional Ethics Committee in the Second Affiliated Hospital of Guangzhou Medical University. 38 patients’ tumor tissues were used for immunohistochemistry. Written informed consents were acquired from each patient relying on guidelines of the Declaration of Helsinki.GPR37  at 4°C for 12 h. The next day, slides were washed, stained with secondary antibodies and 3, 3′-diaminobenzidine, counterstained with hematoxylin, dehydrated and mounted. The sections were reviewed and scored independently by two observers. The results of immunohistochemistry of GPR37 between the different glioma was analyzed by Image Pro Plus image analysis software [Tissue sections were deparaffinised, soaked in TrisEDTA buffer (pH 9.0) boiled in a microwave and then incubated with antibodies against software , take thsoftware .GPR37 expression and other clinicopathological parameters  on the OS and DSS was evaluated using univariate and multivariate cox analysis, with P value < 0.05 as the cut-off criterion. The R package ‘forestplot,’ was used to calculate the P value, HR and 95% CI of each variable.TCGA data were combined and plotted in a matrix. The impact of GPR37 were obtained using the LinkFinder module of LinkedOmics (http://www.linkedomics.org/), and a heat map of the top 50 positively or negatively correlated genes was generated [GPR37 were identified using the STRING database (https://string-db.org) [GPR37high and GPR37low groups, and the differentially expressed genes (DEGs) between the groups were identified using the DESeq2 R (version: 1.26.0) package with P < 0.05 and |log FC|≥1.0 as the thresholds [The gene co-expressed with enerated . The gen-db.org) , and a p-db.org) . The gliresholds . The hubresholds , 23.GPR37high and GPR37low groups [https://www.gsea-msigdb.org/gsea/msigdb/collections.jsp#C2) with a number of size 3 and 10000 simulations [padjust < 0.05 were considered statistically significant.The Clusterprofiler program was used to perform gene set enrichment analysis (GSEA) to identify biological pathways that differed significantly between the w groups , 25. Stuulations . Genes wGPR37 expression with the infiltration of eosinophils, macrophages, NK cells, neutrophils and T cells was analyzed. OS was examined as a function of GPR37 expression, M2 macrophage and cancer-associated fibroblasts (CAFs) in the TIMER2.0 (http://timer.cistrome.org) database [The single-sample GSEA (ssGSEA) was used to quantify the tumor infiltration of 24 immune cell types based on TCGA data using the R GSVA package , 28. Thedatabase .GPR37 expression was investigated using Pearson correlation analysis. Correlation coefficients (R) and Benjamin–Hochberg-adjusted P-values for different methylation sites were obtained. GPR37 methylation and the Kaplan–Meier-based correlation between GPR37 hyper/hypomethylation and OS were visualized using the MethSurv (https://biit.cs.ut.ee/methsurv) program [The relationship between DNA methylation and  program .http://gepia2.cancer-pku.cn/#index), Genotype-Tissue Expression Project , The Cancer Genome Atlas , XENA platform , Human Protein Atlas (https://www.proteinatlas.org/), LinkedOmics database (http://www.linkedomics.org/), STRING database (https://string-db.org), Molecular Signatures database , Tumor Immune Estimation Resource 2.0 database , MethSurv database (https://biit.cs.ut.ee/methsurv).All of the data utilized in this study came from publicly accessible databases. The databases that were used throughout the investigation are listed below. Gene Expression Profiling Interactive Analysis 2 database  (P < 0.001). In addition, immunohistochemical analysis was applied to observe the distribution and protein levels of GPR37. The clinicopathological characteristics of 38 patients are shown in GPR37 expression in different grade of glioma were statistically analyzed in 38 samples. As shown in GPR37 was expressed in neurons and glia and exhibited more elevated expression levels in GBM tissues. Immunohistochemical staining of clinical 38 samples also confirmed that the different level of GPR37 expression in tumor tissues were in CNS WHO grade 2, 3 and 4 of glioma  and GPR37low (n = 352) groups based on the median expression value. As shown in GPR37 in glioma was significantly associated with the CNS WHO grade, histological type,1p/19q codeletion, the IDH status, CDKN2A/B homozygous deletion, age and primary therapy outcome (P < 0.05), while no significant correlation was seen with gender (P > 0.05). Moreover, univariate logistic regression analysis , P < 0.001), histological type , P < 0.001), CDKN2A/B homozygous deletion , P < 0.001), age , P < 0.001).We further analyzed the data from TCGA databases to explore a possible link between analysis  showed aGPR37 expression, grade, histological type, CDKN2A/B homozygous deletion, age, gender and primary therapy outcome using TCGA data indicated that GPR37 was significantly correlated with both OS  and DSS . In addition, multivariate analysis identified GPR37 as an independent risk factor for OS  and DSS . The GPR37high phenotype a significant enrichment of GO terms for positive regulation of macrophage derived foam cell differentiation, negative regulation of T cell receptor signaling pathway, leukocyte proliferation, B cell proliferation and myeloid leukocyte differentiation, whereas immunoglobulin complex, immunoglobulin complex circulating, antigen binding, immunoglobulin receptor binding and humoral immune response mediated by circulating immunoglobulin were significantly enriched in the GPR37low phenotype , T helper cells, T effector memory (Tem) cells (P < 0.001), T central memory (Tcm) cells (P < 0.01), T follicular helper (Tfh) cells, tgd, Th1 cells and activated DCs (aDCs) (P < 0.05). On the other hand, no significant correlation was seen with B cells, cytotoxic cells, DCs, immature DCs (iDCs), mast cells, NK CD56bright cells, Th17 cells, Th2 cells and Treg cells. As shown in GPR37 was positively associated with infiltration levels of Eosinophils , as did higher infiltration of cancer associated fibroblasts  to determine the possible impact of GPR37 expression on the response to immunotherapy. The GPR37high group had high levels of CD274, PDCD1LG2, PDCD1, CD80, CD86, CD28, CTLA4, PVR, TIGIT, CD96, CD226, HAVCR2, LGALS9, CD47, SIPRA, CD200, CD200R1, CIITA, and LAG3 expression  to the cell membrane, metalloproteinases (MPs) can process the N-terminal of GPR37, so that GPR37 can be transformed from precursor form to mature complete glycosylation form and stably exist in the cell membrane in the cleaved form [GPR37 is enriched in apical part of cell, apical plasma membrane, cilium plasm, axoneme etc., has molecular function of passive transmembrane transporter activity, channel activity, substrate−specific channel activity etc., and participates in biological processes such as sensory perception of mechanical stimulus, axon ensheathment, ensheathment of neurons, ciliary movement and so on. In addition to these, KEGG analysis revealed that GPR37 may be involved in regulating ether lipid metabolism, fat digestion and absorption, and histidine metabolism pathways. Ether lipid biosynthesis is unique to the peroxisome and is regulated by the peroxisome proliferator activated receptor (PPAR) [GPR37 was enriched in the PPAR signaling pathway, which is consistent with previous findings. In summary, GPR37 may increase the incidence and development of glioma by regulating the PPAR pathway, which requires more investigation.PPI network analysis further revealed that  studies . GPR37 welopment –47. Furtterminal . It has of GPR37 . During ved form . In our r (PPAR) –54. PPARr (PPAR) . Nwankwor (PPAR) , 57. PPAr (PPAR) . We founGPR37 is related to the increased infiltration of eosinophils, macrophages, NK CD56dim cells, NK cells, neutrophils and T cells etc. A number of growth factors and cytokines can be released by tumor associated macrophages (TAMs), which are drawn into the glioma environment, have immunological capabilities, and are able to react to the growth factors produced by cancer cells [GPR37 was significantly associated with the infiltration of macrophages, which are the most prominent inflammatory cells in tumor tissues [IL-10 and TGFB1 [GPR37 in macrophages facilitates phagocytosis and the regression of inflammatory pain [GPR37, the receptor of specialized pro-resolving mediators (SPMs), can bind to neuroprotectin D1 (NPD1) to inhibit the proinflammatory cytokine IL-1 and increase production of IL-10 and TGFB1. This polarizes the macrophages to the M2 phenotype and relieves inflammatory pain [GPR37 was positively correlated with M2-like TAM markers, and higher proportion of M2 macrophages predicted worse prognosis in the GPR37high patients. Thus, GPR37 maybe contribute to glioma progression by recruiting TAMs and promoting M2 polarization.The tumor immune microenvironment consists of various immune and inflammatory cells that play an important role in tumor development and progression . In thiser cells . TAMs en tissues . The tum tissues , 63. Glind TGFB1 . Furtherory pain . Bang anory pain , 67. ConGPR37 was positively correlated with several ICPs such as CD274, PDCD1LG2, PDCD1, CD80, CD86, CTLA4, PVR, TIGIT, CD96, CD226, HAVCR2, LGALS9, CD47, SIPRA, CD200, CD200R1, CIITA and LAG3, indicating that GPR37 is a promising immune-related gene that can influence immunotherapy response in glioma patients.Immune checkpoint inhibitors (ICIs) can effectively eliminate tumor cells by activating the anti-tumor immune responses , 69. ICPGPR37 promoter was associated with increased expression, and this result was more obvious in LGG than in GBM. Furthermore, LGG patients with hypermethylated promoter regions showed better prognosis. Therefore, the impact of specific GPR37 methylation sites on gene expression and mortality, particularly in LGG patients, needs further investigation.DNA methylation is one of the variables of tumor growth , 75. ProGPR37 is frequently overexpressed in glioma and is an independent prognostic predictor. It is involved in peroxisome control via the PPAR pathway, and is also associated with the infiltration of M2 macrophages and other immune cells. What is more, the overexpression of GPR37 in glioma may be due to DNA hypomethylation. Our findings provide new insights into the possible mechanisms of glioma progression, particularly in the context of the tumor immune environment, which can help develop individualized treatment.Supplementary Figure 1Supplementary Table 1Supplementary Tables 2-5


Diffuse large B-cell lymphoma (DLBCL) and primary mediastinal B-cell lymphoma (PMBCL) are aggressive histological subtypes of non-Hodgkin’s lymphoma. Improved understanding of the underlying molecular pathogenesis has led to new classification and risk stratification tools, including the development of cell-free biomarkers through liquid biopsies. The goal of this study was to investigate cell-free RNA (cfRNA) biomarkers in DLBCL and PMBCL patients.Blood plasma samples (n=168) and matched diagnostic formalin-fixed paraffin-embedded (FFPE) tissue samples (n=69) of DLBCL patients, PMBCL patients and healthy controls were collected between 2016-2021. Plasma samples were collected at diagnosis, at interim evaluation, after treatment, and in case of refractory or relapsed disease. RNA was extracted from 200 µl plasma using the miRNeasy serum/plasma kit and from FFPE tissue using the miRNeasy FFPE kit. RNA was subsequently sequenced on a NovaSeq 6000 instrument using the SMARTer Stranded Total RNA-seq pico v3 library preparation kit.BECN1, PRKCB, COPA, TSC22D3, MAP2K3, UQCRHL, PTMAP4, EHD1, NAP1L1 pseudogene) and a 5-gene signature  were significantly associated with inferior progression-free and overall survival in DLBCL patients, respectively, independent of the NCCN-IPI score.Higher cfRNA concentrations were demonstrated in lymphoma patients compared to healthy controls. A large number of differentially abundant genes were identified between the cell-free transcriptomes of DLBCL patients, PMBCL patients, and healthy controls. Overlap analyses with matched FFPE samples showed that blood plasma has a unique transcriptomic profile that significantly differs from that of the tumor tissue. As a good concordance between tissue-derived gene expression and the immunohistochemistry Hans algorithm for cell-of-origin (COO) classification was demonstrated in the FFPE samples, but not in the plasma samples, a 64-gene cfRNA classifier was developed that can accurately determine COO in plasma. High plasma levels of a 9-gene signature (Total RNA sequencing of blood plasma samples allows the analysis of the cell-free transcriptome in DLBCL and PMBCL patients and demonstrates its unexplored potential in identifying diagnostic, cell-of-origin, and prognostic cfRNA biomarkers. DLBCL can occur ymphomas . AlthougTwo decades ago, gene expression profiling (GEP) was used to discriminate between different cell-of-origin (COO) DLBCL subgroups, germinal center B-cell–like (GCB) and activated B-cell–like (ABC), which was clinically implemented through the use of surrogate immunohistochemistry (IHC) algorithms , 4. SincA liquid biopsy is the process of investigating tumor-derived cells or biomaterials like cell-free nucleic acids, metabolites, proteins, or extracellular vesicles through biofluid sampling, without the need of a tissue biopsy. Potential advantages include its non-invasive nature, its ability to reflect inter- and intra-tumor heterogeneity, and the possibility of repeated measurements through longitudinal profiling during disease course or treatment . Althoug22.12.1.1in situ hybridization (FISH) was performed for MYC and BCL2 rearrangements to identify DHL. Therapy response was assessed by F-fluorodeoxyglucose Positron Emission Tomography/computerized tomography (FDG-PET/CT) according to the Lugano guidelines  and 14 PMBCL patients were collected during the period 06/2016 and 09/2021 at Ghent University Hospital in Ghent (Belgium) and AZ Delta hospital in Roeselare (Belgium). Plasma samples were drawn at the time of diagnosis, at interim evaluation (after 4 cycles of immunochemotherapy), at final evaluation, and in the case of R/R disease. An overview of the collected plasma samples at each timepoint can be found in 2.1.2Plasma was obtained by using a one-step centrifugation protocol (1900g x 15min without brake at room temperature) and subsequently frozen and stored at -80°C. FFPE samples were obtained from the Pathology Department of Ghent University Hospital and were re-examined by an experienced pathologist to confirm the correct diagnosis, to determine the percentage of tumor invasion, and to select adequate tumor blocks. Five scrolls of 10 µm were cut from FFPE blocks on a Leica RM2125 RTS manual rotary microtome  and put into RNase-free Eppendorf tubes . If the FFPE also included non-malignant tissue, macrodissection was performed to enrich lymphoma invaded tissue. The FFPE scrolls were processed within 4 hours after preparation.2.1.3RNA extraction was performed on 200 µl of plasma and on 5 scrolls of 10 µm using the miRNeasy serum/plasma kit and miRNeasy FFPE kit, respectively. In each plasma sample, 2 µL of Sequin spike-in controls were added to the sample lysates , as well as 2 µL of External RNA Control Consortium (ERCC) spike-in controls  were added to the RNA eluate. Genomic DNA was removed by adding 1 μL HL-dsDNase  and 1.6 µL reaction buffer  to 12 µL RNA eluate, followed by 10 min incubation at 37°C, and 10 min incubation at 55°C to inactivate the DNase. RNA was subsequently stored at -80°C and thawed on ice immediately before library preparation.2.1.4Total RNA sequencing libraries were prepared starting from 8 µL of RNA eluate (plasma) or 40 ng total RNA (FFPE) using the SMARTer Stranded Total RNA-Seq Kit v3 – Pico Input Mammalian  according to the manufacturer’s protocol. Equimolar library pools were prepared based on qPCR quantification with KAPA Library Quantification Kit . The libraries were paired-end sequenced (2x100 nucleotides) on a NovaSeq 6000 instrument using a NovaSeq S2 kit  with standard workflow loading of 0.65 nM (2% PhiX). BCL files generated by the Illumina sequencing system were processed using the Illumina Bcl2fastq (v. 2.20) software to generate and demultiplex fastq files. Raw reads were assessed for quality using FastQC (v. 0.11.9) . Next, t2.22.2.1Blood plasma RNA concentration was determined as previously described . Briefly2.2.2Differential abundance analysis on normalized counts was performed using DESeq2 (v. 1.36.0) . Counts 2.2.3To investigate the performance of a tissue-derived GEP to correctly classify FFPE and plasma samples based on COO as predicted by IHC, both a normalized rank and a standardized abundance procedure were applied  . Subsequently, a hierarchical clustering was performed on the selected genes using a Euclidean distance and average agglomeration method (“stats” R library v. 3.6.2). Next, the clusters were detected using the Dynamic Tree Cut (v. 1.63-1) R library to avoid the arbitrary choice of the dissimilarity cut-off between clusters when performing a fixed height tree cut . For eac 0.7-25) . Progres2.2.52 transformed fold changes between the different groups obtained from DESeq2 differential abundance analysis  was performed to explore the functionally enriched pathways and hallmark gene sets related to subgroups, based on the loganalysis . Signifianalysis . Pathway2.2.6All analyses were conducted using the R statistical software package (v. 4.0.5) . Kruskal33.1A total of 41 DLBCL and 14 PMBCL patients were included. The mean age at inclusion was 65.3 and 37.0 years, respectively Table 1.The DLBCL patients were treated with first-line R-(mini)CHOP and the PMBCL patients with dose-intensive rituximab, doxorubicin, cyclophosphamide, vindesine, bleomycin, and prednisone (R-ACVBP) with subsequent consolidation or dose-adjusted EPOCH-rituximab therapy (DA-EPOCH-R) , 39. Aft3.2The blood plasma cfRNA concentration was higher in the lymphoma patients at the time of diagnosis compared to healthy controls  signaling, Tumor Necrosis Factor (TNF) alpha signaling via Nuclear factor kappa-light-chain-enhancer of activated B-cells (NF-κB), epithelial mesenchymal transition, inflammatory response, and organization/degradation of the extracellular matrix (ECM). Moreover, compared to PMBCL samples, DLBCL samples showed enrichment for oxidative phosphorylation, KRAS signaling, G2M checkpoint signaling, NOTCH signaling, stabilization of p53, and regulation of Phosphatase and Tensin homolog (PTEN) stability and activity. Compared to the DLBCL samples, the PMBCL samples demonstrated enrichment for coagulation, epithelial mesenchymal transition, MAPK activation, as well as ECM organization and regulation. , for PMBCL versus controls , and for PMBCL versus DLBCL .To investigate the differences between the plasma and tissue compartment, the overlap was assessed between the DAG obtained in the diagnostic plasma samples and the matched FFPE samples. 3 A totaCD19, CD20, PAX5, IRF4, BCL6), T-cell differentiation , thymic B-cell differentiation , NF-κB , cell proliferation (MKI67), activation (CD30), and apoptosis (BCL2). As expected, many of these genes were differentially abundant between the lymphoma subtypes and healthy controls in tissue samples. These differences were, however, not significant for most markers in the matched plasma samples. Moreover, when comparing DLBCL to control samples, discordant results were seen for PAX5  and BCL2  compared to the findings in the FFPE samples. When comparing PMBCL to control samples, concordant results were seen for BCL2 (p=0.0025) and CD3 (p=0.0018) and discordant results were noted for CD20 (p=0.0095), IRF4 (p=0.0011), and PAX5 (p=0.041). . 8.Lastly, a differential abundance analysis was performed for cfRNA biomarkers that were previously reported in liquid biopsy studies involving DLBCL and/or PMBCL patients –45. For 3.3.2In clinical practice, IHC markers are used to discern the COO subtypes of DLBCL , as well as DEL status (MYC and BCL2) , 9. To cNext, we checked if a tissue-derived GEP, containing the best COO class-predicting genes as initially identified in the Lymphochip microarray data by Alizadeh et al., could correctly classify the DLBCL samples  . When apLastly, no DAG could be identified between DEL and non-DEL samples, illustrating that the transcriptional differences of these subgroups are not clearly reflected in the plasma compartment. This is in line with the observation of only 3 DAG between the matched FFPE samples.  was significantly associated with decreased PFS, both in univariate (p<0.001) and in multivariable regression analysis (p<0.001), the latter independent of the NCCN-IPI score.  was significantly associated with worse OS in both univariate (p<0.001) and multivariable regression analysis (p=0.004). , PAX5, IGHD, FCRL1, CD79A, TNFRSF13C, IGHM, FCER2, CD72, CD22, CARD11, IRF4, EBF1, BLK, BLNK, STAP1, FAM129C, BACH2, TCL1A, POU2AF1 and FOXP1. Four of these DAG  were also upregulated at pre-therapy timepoint compared to control samples, but decreased significantly during therapy below healthy control levels, with the opposite being true for IGF2. When analyzing the interim time point during therapy, a clear decrease in the aforementioned B-cell related markers was already noted to similar levels as the post-therapy time point. Additionally, a transient increase in neutrophil and inflammation associated markers, including CD177, MMP8, CEACAM8, OLFM4, BPI, MS4A3, and MPO, was demonstrated at the interim timepoint. In contrast, within the DLBCL non-responders, only a transient decrease of several B-cell related markers  was noted when comparing the diagnostic samples to the interim samples, no DAG could be identified between the diagnostic and the R/R samples, and no transient increase in inflammation associated markers was noted.When comparing matched pre- and post-therapy samples in DLBCL responder patients after successful R-CHOP therapy, a total of 184 DAG were identified.  and B-cell lymphoma 2 (BCL2) plasma levels were shown in DLBCL patients compared to healthy controls  and Fibronectin 1 (FN1) levels were not differentially abundant in the DLBCL plasma samples as opposed to control samples, although our results revealed higher levels in the FFPE samples (GAS5), as well as increased Taurine Up-Regulated 1 (TUG1) and increased X-inactive specific transcript (XIST) plasma levels have been demonstrated in DLBCL compared to control plasma samples , has been reported as downregulated in DLBCL patients , Prothymosin Alpha (PTMA), and Nucleosome Assembly Protein 1 Like 1 (NAP1L1), respectively, which have been found dysregulated in various malignancies ((PRKCB) is an integral component of signaling via the B-cell receptor in DLBCL, activating NF-κB and VEGF–mediated angiogenesis, and its tissue expression was shown to be an independent predictor of poor OS (ATF4) levels have been associated with improved survival probability in DLBCL patients , PFS, and OS in DLBCL patients , 58, 66.gnancies –79. Pseugnancies . Protein poor OS , 69. Decpatients . A recenc target .PET-guided treatment of DLBCL remains debatable as direct evidence of improved patient outcomes is still lacking. Outside of a clinical trial, biopsy confirmation of an abnormal interim PET-CT scan is still recommended before switching therapy –83. In rOur study has several limitations. Our findings have been compared with previous studies that have mainly used RT-qPCR or microarray-based abundance analyses and have collected and processed samples using other blood collection tubes and protocols, which may hamper direct comparison of results. Moreover, specific genes can be differentially enriched in EVs compared to the circulating-free compartment, underlining that caution should be applied when comparing the results of studies investigating different biofluid fractions , 88. TheIn conclusion, we present the first study to longitudinally explore the total cell-free transcriptome in DLBCL and PMBCL patients using blood plasma samples. A large number of differentially abundant genes were identified and compared against previous cfRNA biomarker studies. Overall, overlap analyses with matched FFPE samples showed that blood plasma has a unique transcriptomic profile, that significantly differs from that of the tissue. Our results demonstrated a good concordance between tissue-derived gene expression and the Hans algorithm for COO classification in FFPE samples, but not in the plasma samples. Therefore, a cfRNA classifier was developed that can accurately determine COO in plasma. Lastly, high plasma levels of a 9-gene signature and a 5-gene signature were associated with inferior PFS and OS in DLBCL patients, respectively, independent of the NCCN-IPI score. Our results may serve as a reference point for future cfRNA studies, and the biomarkers identified may represent potential targets for in-depth functional analysis.https://ega-archive.org, ID EGAS0000100758 (dataset EGAD00001011679).The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: The studies involving humans were approved by medical Ethical Committee of Ghent University Hospital, Ghent, Belgium (EC/2016/0307). The studies were conducted in accordance with the local legislation and institutional requirements. The participants provided their written informed consent to participate in this study.PD, FO, and JV performed study concept and design; PD, EG, JV, and FO performed writing, review, and revision of the paper; PD, EG, JV, and FO provided acquisition, analysis and interpretation of data. EG and PD performed statistical analysis; JVD, DD, JA, KV, MVdL, and KDG provided technical and material support. All authors contributed to the article and approved the submitted version.


Survival, body weight, and wound healing were tracked for 30 days, and mouse brain samples were collected on day 30 after SW, RI, RCI, and the sham control. Our results showed that RCI caused more severe animal death and body weight loss compared with RI, and skin wound healing was significantly delayed by RCI compared to SW. RCI and RI increased the chemokines Eotaxin, IP-10, MIG, 6Ckine/Exodus2, MCP-5, and TIMP-1 in the brain compared to SW and the sham control mice, and the Western blot results showed that IP-10 and p21 were significantly upregulated in brain cells post-RI or -RCI. RI and RCI activated both astrocytes and endothelial cells in the mouse brain, subsequently inducing blood–brain barrier (BBB) leakage, as shown by the increased ICAM1 and GFAP proteins in the brain and GFAP in the serum. The Doublecortin (DCX) protein, the “gold standard” for measuring neurogenesis, was significantly downregulated by RI and RCI compared with the sham group. Furthermore, RI and RCI decreased the expression of the neural stem cell marker E-cadherin, the intermediate progenitor marker MASH1, the immature neuron cell marker NeuroD1, and the mature neuron cell marker NeuN, indicating neural cell damage in all development stages after RI and RCI. Immunohistochemistry (IHC) staining further confirmed the significant loss of neural cells in RCI. Our data demonstrated that RI and RCI induced brain injury through inflammatory pathways, and RCI exacerbated neural cell damage more than RI.Radiation injury- and radiation combined with skin injury-induced inflammatory responses in the mouse brain were evaluated in this study. Female B6D2F1/J mice were subjected to a sham, a skin wound (SW), 9.5 Gy  The risk of radiation-induced injury has increased due to the expanding of nuclear proliferation, terrorist activities, and the distribution of radioactive materials. Radiation combined injury (RCI), which is radiation exposure coupled with other forms of injury, such as wounds, burns, blasts, trauma, hemorrhage, and/or sepsis, results in more than 60% of injuries after a nuclear explosion . RCI greRadiation-induced massive proinflammatory cytokine release (cytokine storm) from different organs and tissues has been reported by many research groups, including ours ,7,8,9. RRadiation-induced brain injury in humans is described in terms of acute injury, early-delayed injury, and late-delayed injury . Acute bp = 0.76; N = 20). However, RCI caused significantly more animal death than RI on day 16 (p = 0.047). SW did not cause any animal death. Body weight was a critical risk indicator of irradiation and was significantly reduced by RI and RCI at all time points, whereas SW did not cause body weight loss compared with the sham-irradiated mice. The body weight in the RCI group of mice further dropped on days 3, 7, 10, and 14 post-TBI and was significantly lower than in the RI group of mice, as shown in p < 0.05). Furthermore, skin wound healing was measured in the SW and RCI groups. Skin wounds were completely closed on day 14 in the SW group; however, RCI significantly delayed wound healing at all time points, and wounds were not closed on day 28, as shown in Mice receiving 9.5 Gy TBI (RI), RI combined with a skin wound (RCI), and a skin wound without irradiation (SW) were monitored for 30 days, and the percentages of survival on day 30 after insults were calculated. Body weight and skin wound healing were measured on days 0, 3, 7, 10, 14, 21, 24, and 28 post-RI, -RCI and -SW; the data are summarized in The dysregulation of cytokines and chemokines is a central feature in the development of neuroinflammation. To evaluate the RI- and RCI-induced inflammatory responses, proinflammatory cytokine and chemokine levels in mouse brains were analyzed. Brain tissues were collected from SW, sham-irradiated, RI, or RCI mice 30 days after TBI. The collected brain tissue lysates were subjected to a cytokine antibody array analysis by Eve Technologies  as described in the Materials and Methods section, with each group including 4–5 mice (N = 4–5). Forty-four cytokines/chemokines were analyzed, and the results indicated that six chemokines were increased in samples from the RI and/or RCI groups, whereas no cytokine/chemokine change was observed in the SW mouse brains compared with those samples from the sham control group. The data are summarized in To further confirm the RI- and RCI-induced inflammation and toxicity in mouse brain cells, we examined the IP-10 expression in the mouse brain samples since this chemokine is expressed by neural cells, glia, and stromal cells and plays a critical role in controlling the entry of leukocyte subsets into the brain after injury . An immuRadiation-induced inflammatory response in the mouse brain could cause microglial, endothelial cell, and astrocyte activation and subsequently result in neurovascular structure damage, as well as BBB and BCSFB leakage and dysfunction. To evaluate the function of the BBB in the mouse brain after a skin wound, RI, and RCI, the microglial activation marker MHC (Major histocompatibility complex)-II, the astrocyte activation marker GFAP , and the neuroendothelial cell activation marker ICAM1 (Intercellular adhesion molecule-1) were examined in mouse brain samples. The representative immunoblotting results are shown in p < 0.01). The RCI group had the highest proportion of GFAP-positive staining compared with the other groups. ICAM1 expression in mouse brain tissues from the different groups was also examined using IHC staining, and images of the slides from a representative mouse brain tissue are shown in p < 0.01). The RCI group had the highest proportion of ICAM1-positive staining, with bigger cell sizes than the other groups.The expressions of GFAP and ICAM1 in hindbrain tissue were also confirmed via immunohistochemistry (IHC) staining as described in the Materials and Methods section. Sample slides from three mice/group were stained with specific antibodies, and two slides from each mouse were counted. The images in Furthermore, the levels of ICAM1 and GFAP in the mouse brain and serum from the sham-irradiated control, wound alone, RI, and RCI mice were measured via ELISA (Doublecortin (DCX) is a determinant factor in brain growth that is expressed in various regions of the developing nervous system. To evaluate the toxicity of RI and RCI in mouse neuron cells, we determined the DCX levels in mouse brains using ELISA (N = 4–5 per group), and Next, the different stages of neural cell population were evaluated via immunoblotting assay and IHC staining using specific antibodies, including anti-E Cadherin , anti-MASH1 (a progenitor cell maker), anti-NeuroD1  and anti-NeuN , as described in the Materials and Methods section. RI and RCI decreased neural cell levels at all stages, as shown in the Western blot data b. These Our previous research demonstrated that radiation combined injury (RCI) leads to higher mouse mortality rates, delayed wound healing, and organ dysfunction compared to radiation injury (RI) or physical trauma alone . In addiWe measured the levels of 44 cytokines/chemokines in the brains of mice subjected to RI, RCI, skin wound (SW), or sham-irradiation, and compared the levels between the groups using statistical analysis. Our results showed that, 30 days after RI and RCI, 6 out of 44 cytokines/chemokines levels were significantly elevated in the mouse brains compared with samples from sham-irradiated control. All six elevated proinflammatory factors were chemokines, including Eotaxin, IP-10, MIG, MCP-5, and TIMP-1, as shown in To further understand the inflammatory signaling in RI- and RCI-induced mouse brain injury, we examined the major senescence/apoptosis regulator p16, p53, phosphorylated p53 (ser 15), and p21 in mouse brains. Our data demonstrated that p21 was significantly upregulated in mouse brains 30 days after RI and RCI, indicating a potential role of p21 in RI- and RCI-induced brain injury. Since p21 has multiple functions that largely depend on direct p21 signal network interactions and the time , furtherOur results also showed that RI and RCI were highly upregulated the astrocytes activation marker GFAP and the endothelial cell activation marker ICAM1 in all brain tissues from the left- and right-side brain and the hindbrain . GFAP isFinally, RI and RCI inhibited neurogenesis, as shown by the reduction in DCX protein and damaged neural cells in all developing stages. DCX is a determinant factor in the growing brain and is expressed in various regions of the developing nervous system with cell proliferation during neurogenesis and neuronal migration . Our datAnimals were housed in an Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC)-approved facility at the Uniformed Services University of the Health Sciences (USUHS), Bethesda, MD, USA, and all animal-handling procedures were performed in compliance with guidelines of the National Research Council (2011). All animal experiments were reviewed and approved by the USUHS Institutional Animal Care and Use Committee (IACUC protocol # AFR-20-021). All procedures and animal handling were carried out in accordance with the USUHS Department for Laboratory Animal Resources (DLAR) guidelines.B6D2F1/J female mice (12–13 weeks old) were received from Jackson Laboratory  : wound area = π × A/2 × B/2 (A and B represent diameters at right angles to each other).Mice receiving 9.5 Gy TBI (RI), RI combined with skin wound (RCI), and skin wound without irradiation (SW) were closely monitored for 30 days as indicated in a previous report . Morbid Mouse brain samples were collected from euthanized animals 30 days after sham-irradiation, SW, RI, or RCI (N = 4–6/group). Briefly, the whole brain was first separated into forebrain (right and left side) and hindbrain pieces with a razor blade, and then, stored at −80 °C until further use.g, the supernatant was collected and protein concentrations were determined using a BCA assay kit . The collected supernatants (brain tissue lysates) were subjected to a cytokine antibody array analysis by Eve Technologies Company . In brief, 100 µL of brain tissue lysate at a concentration of 4 µg/µL was subjected to mouse cytokine/chemokine 44-plex discovery assay® array (MD44). A total of 44 cytokines/chemokines, including Eotaxin, Erythropoietin, 6Ckine, Fractalkine, G-CSF, GM-CSF, IFNB1, IFNγ, IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-11, IL-12p40, IL-12p70, IL-13, IL-15, IL-16, IL-17, IL-20, IP-10, KC, LIF, LIX, MCP-1, MCP-5, M-CSF, MDC, MIG, MIP-1α, MIP-1β, MIP-2, MIP-3α, MIP-3B, RANTES, TARC, TIMP-1, TNFα, and VEGF-A, were measured.The frozen mouse brain tissues were homogenized in RIPA lysis and extract buffer  supplemented with a HaltTM protease inhibitor Cocktail  using the Stomachert 80 Biomaster Lab System  following the manufacturer’s recommendations. After 15 min centrifugation at 12,000× g, the supernatant was collected and protein concentrations were determined using a BCA assay kit . The collected homogenates were denatured in Laemmli buffer and loaded for SDS-PAGE electrophoresis. Following the standard procedures, immunoblotting was then performed using an enhanced chemiluminescence kit . The images were captured using a ChemiDoc MP Imaging System . Antibodies for GFAP , ICAM 1 , IP10 , p21 , p53 , MASH 1 , E-cadherin , NeuroD 1 , and NeuN  were purchased from Abcam , MHC-II  from Santa Cruz Biotechnology, p16  and from Invitrogen . β-actin  was obtained from Sigma-Aldrich .The frozen mouse brain tissues were homogenized in N-PER Neuronal Protein Extraction Reagent  supplemented with a protease inhibitor tablet using a tissue homogenizer , following manufacturer’s recommendations. After 15 min centrifugation at 12,000× Western blots were stripped using a Restore™ PLUS Western Blot Stripping Buffer  for 30 min at room temperature, followed by thorough washing with TBST (3× for 15 min each) prior to re-probing the blot with different antibodies.The mouse brain samples were fixed in 10% formalin, embedded in paraffin, and cut into 5 µm sections per slide. IHC staining was conducted via antigen retrieval in a citrate buffer (pH 6.0) at 100 °C for 20 min, followed by gradual cooling to room temperature. After quenching the endogenous peroxidase activity using BloxAll solution , the specimens were blocked with 2.5% normal serum , and then, incubated at 4 °C overnight with primary antibodies . The specimens were washed and subsequently incubated using ImmPRESS HRP IgG Polymer Detection Kits , and the ImmPACT DAB Kit  was used to develop the substrate to the desired stain intensity. The slides were counterstained with hematoxylin, and images were scanned using a Zeiss Axio Scan.Z1 system.The IHC-positive staining was quantified using ImageJ Fiji software according to our published protocol . All theGFAP and ICAM1 in mouse brain and serum, and DCX in the brain were quantitated via ELISA. The mouse ELISA kit for GFAP (CSB-E08603m) was purchased from CUSABIO , ICAM1 (ab100688) was purchased from abcam, and DCX (MBS451857) was purchased from MyBioSource. The GFAP, ICAM1, and DXC levels in the serum and/or brain tissue were determined in duplicate following assay instructions provided by the manufacturers (N = 5 mice/group). Briefly, 50 µL/well of mouse serum  or 50 µg protein (for ICAM1), 5µg protein (for GFAP), and 75 µg protein (for DXC) from each brain sample were added to a 96-microwell plate precoated with anti-mouse GFAP, ICAM1, or DXC antibody, respectively. The plates were incubated for 2 h. After removing the liquid, the biotin antibody was added to each well and incubated for 1 h. After washing, the HRP avidin was added to the microwells and incubated for 1 h. After another washing stage, the substrate reagent was added to each well and incubated for 30 min. An acid solution was added to each microwell to terminate the enzyme reaction and stabilize the color development. The optical density (O.D.) of each microwell was measured at 450 nm using a microplate reader. The minimum detection limit for mouse GFAP was 3.12 pg/mL, for ICAM1 was 24.69 pg/mL, and for DXC was 0.156 ng/mL.p < 0.05 was taken as statistically significant. For other cell biology data, differences between means were compared via analysis of variance (ANOVA) and Student’s t tests. p < 0.05 was considered statistically significant. Results are presented as means ± standard deviation (SD) or standard error of the mean (SEM).The 30-day survival of mice was analyzed using Kaplan–Meier analysis. Fisher’s exact test was used to compare survival among groups at the end of 30 days after TBI, and Previous studies have suggested that radiation-induced brain injury only occurs at very high doses of radiation exposure . However


Chimonanthus praecox (L.) Link, Calycanthaceae) is an esteemed ornamental flowering shrub known for its distinct blooming period in winter, vibrant color petals, and captivating floral fragrance. Basic helix-loop-helix (bHLH) transcription factors (TFs) play pivotal roles as key regulators in secondary metabolites biosynthesis, growth, and development in plants. However, the systematic analysis of the bHLH family members and their role in the regulation of floral traits in Wintersweet remains insufficiently understood. To bridge this knowledge gap, we conducted a comprehensive genome-wide analysis of the C. praecox bHLH (CpbHLH) gene family, identifying a total of 131 CpbHLH genes across 11 chromosomes. Phylogenetic analysis classified these CpbHLH genes into 23 subfamilies, wherein most members within the same subfamily exhibited analogous intron/exon patterns and motif composition. Moreover, the expansion of the CpbHLH gene family was primarily driven by segmental duplication, with duplicated gene pairs experiencing purifying selection during evolution. Transcriptomic analysis revealed diverse expression patterns of CpbHLH genes in various tissues and distinct stages of Wintersweet flower development, thereby suggesting their involvement in a diverse array of physiological processes. Furthermore, yeast 2-hybrid assay demonstrated interaction between CpbHLH25 and CpbHLH59  as well as with CpbHLH112 and CpMYB2, suggesting potential coordinately regulation of secondary metabolites biosynthesis in Wintersweet flowers. Collectively, our comprehensive analysis provides valuable insights into the structural attributes, evolutionary dynamics, and expression profiles of the CpbHLH gene family, laying a solid foundation for further explorations of the multifaceted physiological and molecular roles of bHLH TFs in Wintersweet.Wintersweet ( Arabidopsis thaliana (L.) Heynh. (Brassicaceae), certain atypical bHLHs have been identified and characterized, featuring a less conserved basic region that is essential for DNA binding [Transcription factors (TFs) are pivotal regulators that orchestrate a wide range of biological processes using the activation or repression of target gene expression ,2,3,4,5. binding ,15.Vaccinium spp.), VcbHLHL1 stimulates anthocyanin accumulation and pigment development by interacting with VcMYBL1 and VcWDL2 [AtTT8, AtGL3, and AtEGL3 in A. thaliana [DcTT8 in Dendrobium candidum [A. thaliana, the HEC gene encoding bHLH TF was found to be involved in female reproductive tract development, depending on the level of loss in HEC function, infertility, and developmental abnormalities observed in plants [AabHLH112 as a positive regulator of sesquiterpenes biosynthesis  in Artemisia annua L. (Asteraceae) [Solanum lycopersicum L. (Solanaceae), knockdown of SlMYC1 resulted in the reduction in monoterpene content in leaf and stem trichome and sesquiterpenes (β-caryophyllene and α-humulene) in leaf trichome, while the content of β-caryophyllene and α-humulene increased in stem trichome, indicating differential regulation by SlMYC1 [The bHLH TFs are important regulators of various biological processes in plants, including secondary metabolism ,17,18, pd VcWDL2 . Severalthaliana  and DcTTcandidum . In A. tn plants . BIGPETAn plants . Recent eraceae) . In Solay SlMYC1 . Given tChimonanthus praecox, commonly known as Wintersweet, has a long history of cultivation in China, dating back over a thousand years [CpbHLH1 [CpTT8 [CpbHLH13, and CpMYC2 [CpbHLH TFs, including evolutionary history, chromosomal distribution, gene duplication, protein motifs, and gene structures. Furthermore, this study conducted an analysis of the expression patterns of CpbHLH genes using transcriptome data obtained from different organs of Wintersweet plants, as well as during various stages of flower development in two Wintersweet genotypes. The findings from this study provide a valuable resource for future investigations exploring the intricate relationship between CpbHLH genes and diverse floral characteristics within Wintersweet.nd years ,35. It ind years . Notablynd years ,37. Addind years ,39. The [CpbHLH1 , CpTT8 [1 [CpTT8 , CpbHLH1d CpMYC2 . To shedd CpMYC2 . This stCpbHLH genes were identified by combining the results of conserved domains and HMM identification. These CpbHLH genes were renamed CpbHLH1 to CpbHLH131 based on their distribution across different chromosomes. Next, we analyzed the physicochemical characteristics of the putative CpbHLHs and found diversities in open reading frames (ORFs), protein length, molecular weight (Mw), isoelectric point (PI), subcellular localization, and GRAVY. Specifically, the ORFs of the CpbHLH genes ranged from 273 to 2277 bp, and their corresponding protein products varied in length from 90 aa to 758 aa. The molecular weight (MW) and isoelectric point (PI) of CpbHLHs were found to range from 10218.55 to 82809.87 and 4.63 to 11.24, respectively , followed by chromosome 4 with 17 CpbHLH genes, while chromosome 11 had the lowest number of CpbHLH genes (4)  . The cluenes (4) . In totaenes (4) .CpbHLHs, a NJ phylogenetic tree was generated by aligning the full-length protein sequences of 131 CpbHLHs, 157 AtbHLHs and six functionally characterized bHLHs . Based on the classification system of the A. thaliana bHLH family [CpbHLHs, including Ia, Ib1, Ib2, II, III(a+c), IIIb, III(d+e), IIIf, IVa, IVb, IVc, IVd, Va, Vb, VII(a+b), VIIIa, VIIIb, VIIIc, IX, X, XI, XII, and XV. None of the CpbHLH genes clustered in the XIII and XIV subfamilies. The largest number of CpbHLH genes (16) was found in the XII subfamily, while the II and VIIIa subfamilies had the fewest number of CpbHLH genes, with only one CpbHLH gene in each  and introns (9) among all CpbHLH genes , light-responsive elements , hormone-responsive elements , stress-related elements , MYB-related elements, MYC elements, Binding site elements, and promoter-related elements. The presence of MRE, MBS, and MBS1 cis-acting elements in the promoter region of CpbHLH genes suggests that MYB TFs may play a role in regulating the transcription of bHLH genes in Wintersweet, potentially modulating the expression of downstream genes  to examine gene duplication events in C. praecox. A total of 61 gene duplication events were identified, resulting in the formation of gene pairs within the CpbHLHs. Among these, five pairs (comprising 8 CpbHLH genes) were found to have arisen through tandem duplication, which occurred on chromosomes 1, 2, and 3 (CpbHLH genes) appeared to be segmental duplicates distributed across all 11 chromosomes  could not be calculated due to sequence divergence.To gain insight into the expansion mechanism of the 2, and 3 . Additioomosomes A. These  process . The Ka/A. thaliana, P. trichocarpa Torr. and A.Gray ex. Hook. , V. vinifera L. (Vitaceae) (dicotyledon), O. sativa L. (Poaceae) (monocotyledon), and C. praecox, collinearity analysis was carried out to identify orthologous bHLH genes across these species. Our analysis revealed a total of 68 orthologous gene pairs between C. praecox and A. thaliana, 187 pairs between C. praecox and P. trichocarpa, 109 pairs between C. praecox and V. vinifera, and 96 pairs between C. praecox and O. sativa , CpbHLH34, CpbHLH99, CpbHLH69, CpbHLH100, CpbHLH64, CpbHLH95, CpbHLH26, CpbHLH20, CpbHLH21, CpbHLH81, CpbHLH5, CpbHLH93, CpbHLH85, CpbHLH84, CpbHLH83, CpbHLH30, and CpbHLH29 (C. praecox and P. trichocarpa) (To explore the evolutionary relationship among bHLH genes of . sativa B. Among LH genes . Additiohocarpa) .CpbHLH genes in different tissues of Wintersweet, the FPKM values of these genes were extracted from transcriptome data of four distinct organ samples, namely roots, leaves, cotyledon, and flowers. CpbHLH genes with FPKM values below 1 in all tissues were excluded, and the remaining genes were used to generate an expression heatmap. Out of the 131 CpbHLH genes, 94 showed expression in at least one of the four tissues analyzed in the transcriptome database. Cluster analysis was performed, resulting in the classification of the 94 CpbHLH genes into seven distinct groups based on their specific expression profiles  and activation domain (AD) fusion constructs. The yeast cells carrying BD-empty+AD-CpMYB2, BD-empty+AD-CpbHLH25, BD-empty+AD-CpbHLH59, and BD-empty+AD-empty did not exhibit any growth or interaction on SD/-Leu-Trp-His-Ade+3-AT+X-α-gal medium . In contCpbHLH1, CpTT8, CpMYC2, and CpbHLH13) being functionally characterized [A. thaliana (162) [O. sativa (183) [Malus domestica (Suckow) Borkh. (Rosaceae) (175) [Prunus avium L. (Rosaceae) (66) [Carthamus tinctorius L. (Asteraceae) (41) [The bHLH genes have demonstrated their involvement in a diverse range of physiological and biochemical processes  in plants ,45,46,47cterized ,40,41. Tcterized  has provna (162) , O. sativa (183) , and Male) (175)  but highae) (66)  and Cartae) (41) . The varae) (41) ,53,54.Prunus mume (Siebold) Siebold and Zucc. (Rosaceae), 100 PmbHLH genes were categorized into 21 subfamilies, while in Camellia sinensis (L.) Kuntze (Theaceae), 134 CsbHLHs were divided into 17 subfamilies [CpbHLH genes clustered into 23 subfamilies and lacked members in XIII and XIV subfamilies  Gaertn. (Polygonaceae) [CpbHLH suggested that this gene family experienced purifying selection, a clear indication of highly conserved evolution. In the collinearity analysis, we found that CpbHLH genes showed a high level of collinearity with P. trichocarpa bHLH genes, followed by V. vinifera, O. sativa, and A. thaliana, indicating that these bHLH genes probably descended from a common ancestor  ,66. The ancestor B. MoreovCpbHLH genes across various tissues. A total of 94 CpbHLH genes were found to be expressed in at least one tissue, and their expression levels varied greatly across tissues  in the VII(a+b) subfamily (AtbHLH24 regulates organ morphogenesis (carpel development) [CpbHLH114, CpbHLH57, and CpbHLH10 was highest in leaves  . Moreoven leaves A. In thebidopsis , suggestCpbHLH genes during the development of Wintersweet flowers, aiming to gain insights into their roles in Wintersweet flowers. In total, 97 CpbHLH genes were expressed in at least one stage of flower development, with 77 differentially expressed genes and 20 genes showing stable expression across three flower development stages in two Wintersweet genotypes  (Arabidopsis [CpbHLH86 clustered with Cryptochrome interacting bHLHs (CIBs) in subfamily XII, which act as positive regulators in CRY2-mediated flowering time in Arabidopsis [CpbHLHs in a similar process in Wintersweet.Next, we delved deeper into the expression profile of enotypes , indicattersweet . In the II (a+c) . These gbidopsis ,70. Furtbidopsis , suggestCpbHLH118, CpbHLH66, and CpbHLH96 clustered with AtbHLH1, AtbHLH2, AtbHLH12, and AtbHLH42 in IIIf subfamily , while in the HLT015 genotype , their expression significantly upregulated at the open flower stage compared to HLT040 genotype (CpbHLH1 (referred to as CpbHLH118 in this study) and CpTT8 (referred to as CpbHLH96 in this study) act as negative and positive regulators of anthocyanin biosynthesis, respectively [CpbHLH66 shares sequence similarity with CpbHLH118 but lacks the DNA binding region. However, it contains the conserved amino acid residues in helix regions required for dimerization, suggesting that CpbHLH66 may form heterodimers with other transcription factors to enhance or repress their activity. Additionally, CpbHLH87, CpbHLH77, and CpbHLH17 also showed differential expression during flower development and between the genotypes. CpbHLH87 showed a higher expression level at bud stage 2 in the HLT015 compared to the HLT040 genotype. It shares the highest sequence similarity with BIM1 , and previous reports have shown that brassinosteroid, along with other phytohormone signaling, enhances anthocyanin accumulation in plants, potentially regulated using ternary MYB-bHLH-WD transcriptional complexes [CpbHLH genes.ubfamily , which hgenotype B, This somplexes ,76. HoweCpbHLH25 in this study) and MYC2 (referred to as CpbHLH59 in this study) in model plants (Arabidopsis and tobacco) increased the β-caryophyllene and Linalool contents, respectively, a reduction in anthocyanin content was also observed in bHLH13 overexpressing plants [CpANS [CpbHLH112 is a potential candidate for regulating scent biosynthesis in Wintersweet flowers [Compared to other TFs, bHLH TFs exhibit versatile regulatory functions, participating in multiple pathways and acting as co-regulators of gene expression alongside other proteins . Previoug plants . In the s [CpANS . Further flowers . TherefoCpbHLH candidates in the Wintersweet genome database using the hidden Markov Model (HMM) profile of the HLH (PF00010) domain that was obtained from the Pfam database [CpbHLHs as well as possible, genes containing incomplete or atypical bHLH structural domains were also retained for subsequent analysis. The physicochemical characteristics of CpbHLH proteins, such as the numbers of amino acids (aa), isoelectric point (pI), and molecular weights (MW), were determined using the ExPASy (ProtParam) online tool (https://web.expasy.org/protparam/ (accessed on 3 January 2023)). The subcellular location of the CpbHLH protein was predicted using WoLF PSORT (https://www.genscript.com/wolf-psort.html (accessed on 3 January 2023)). Furthermore, the sequences of conserved domains in CpbHLH proteins were visualized and analyzed using the Weblogo3 and Jalview software (version 2.11.2.7).The Wintersweet genome sequences and protein sequences, along with annotation information, were acquired from the Wintersweet genome database . The HMMdatabase . All of database  and SMARdatabase  databasehttps://meme-suite.org/meme/tools/meme (accessed on 8 January 2023)), an online tool, was used to examine the motifs (max. number = 20) of the CpbHLH proteins [MEME SUITE  with default parameters. The neighbor-joining (NJ) phylogenetic tree was constructed using pairwise deletion, p-distance method, and 1000 bootstrap replication in MEGA7. NJ phylogenetic tree of CpbHLHs was constructed using the same parameters as described above. The Newick extension of the MEGA NJ was used as a query to obtain the final figure of the NJ tree using the iTOL. The positions of CpbHLH genes on specific chromosomes were visualized using TBtools software (version v2.001) based on the chromosomal location information of genes obtained from the GFF file of the Wintersweet genome database.The protein sequences of CpbHLH gene from its full-length genomic sequence using TBtools and subjected it to the PlantCARE database for the prediction of putative cis-acting elements [We obtained the 2 kb upstream sequence of the start codon of each elements .C. praecox, and to find the homology of the bHLH genes between C. praecox and the other selected plants , the MCscanx tool embedded in TBtools was used. The Ka/Ks calculator integrated in TBtools was employed to examine the Ka and Ks values of duplicated CpbHLH gene pairs, and the Ka/Ks ratio was calculated to evaluate the selection pressure. The genomic information of A. thaliana, P. trichocarpa, V. vinifera, and O. sativa was downloaded from Phytozome.To detect the gene duplication events of bHLH genes within Two genotypes of Wintersweet (HLT015 and HLT040), grown under natural conditions in Heilongtan Park , were selected. The morphological attributes of these genotypes were as follows: (1) HLT015, light yellow-colored flowers with red inner petals; (2) HLT040, dark yellow-colored flowers with yellow petals. For RNA sequencing, three biological replicates of green buds (Stage 1), buds turning yellow (Stage 2), and open flowers (Stage 3) were collected from both genotypes. For tissue-specific expression analysis, cotyledon, leaf, root, and flower tissues of Wintersweet were collected. All samples were immediately frozen in liquid nitrogen and stored at −80 °C until RNA extraction.CpbHLH candidate genes were retrieved from the transcriptome databases generated from three developmental stages of flower  of two Wintersweet genotypes as well as different organs  of Wintersweet plant. We used TBtools to create heat maps that show the transcript profiles of CpbHLH genes based on the FPKM values.The FPKM (Fragments Per Kilobase per Million mapped reads) values of TM 2X qPCR MasterMix (10 µL), cDNA , forward primer (0.5 µL), reverse primer (0.5 µL) and ddH2O (8 µL) on the Roche LightCyclerR 480 II Real-Time PCR platform. The experiments were conducted in triplicates, and β-actin was used as an internal reference for normalization. Finally, the 2−ΔΔCT method was utilized for the calculation of relative gene expression levels. The primers used in this study are listed in To validate the transcriptome results, we used RT-qPCR to measure the transcription levels of the differentially expressed genes (DEGs) with gene-specific primers. The extraction of total RNA from two Wintersweet genotypes flowers at three developmental stages  was carried out using the EASYspin plant RNA extraction kit  as per manufacturer’s protocol. Subsequently, the integrity and purity of RNA were determined using a 1.5% denaturing agarose gel and a Nanodrop-2000 spectrophotometer, respectively. The reverse transcription of RNA into cDNA was carried out using the cDNA Synthesis SuperMix  according to the manufacturer’s instructions. RT-qPCR was performed in a 20 µL reaction volume comprising BlastaqThe open reading frames (ORFs) of CpbHLH25, CpbHLH59, CpMYB2, and CpbHLH112 were amplified using gene-specific primers and cloned separately into pGBKT7 and pGADT7 vectors to generate bait and prey constructs, respectively. We then co-transformed the respective pairs of recombinant vectors into the AH109 yeast strain and performed the Y2H assay as directed by the manufacturer (Clontech). The transformants (AH109 cells) carrying the pairs of recombinant vectors were cultured on synthetic dropout media SD/-Leu-Trp, and the pairs interactions were evaluated on the SD/-Leu-Trp-His-Ade + 3-amino-1,2,4-triazole (3-AT) (40 mM/L) + X-α-gal (25 mg/L) media.CpbHLH genes distributed across the 11 chromosomes of Wintersweet, which were classified into 23 distinct subfamilies. The accuracy of this classification was further supported using motif and gene structure analyses. Notably, gene duplication, particularly segmental duplication, emerged as a key driver of bHLH gene diversity. RNA-seq analysis illuminated distinct expression patterns in various tissues and during flower development, highlighting intricate transcriptional regulation. Furthermore, CpbHLH25 and CpbHLH59  form heterodimers not only with each other but also with CpbHLH112 and CpMYB2, suggesting their potential coordination in regulating secondary metabolite biosynthesis. Overall, these findings offer valuable insights for future research investigating the biological functions of bHLH gene family members in Wintersweet flowers.In the current study, we performed a comprehensive genome-wide analysis of the bHLH gene family in Wintersweet. Our analysis revealed the presence of 131


Cryptococcus neoformans, FEMS Yeast Research, Volume 20, Issue 3, 11 May 2020, https://doi.org/10.1093/femsyr/foaa015.This is a retraction of Jiu Yi, Junjun Sang, Jingyu Zhao, Lei Gao, Yali Yang, Lei Yan, Chao Zhang, Weihua Pan, Guizhen Wang, Wanqing Liao, Transcription factor Liv4 is required for growth and pathogenesis of https://pubpeer.com/publications/4D828B96E005A6D0D0A2A74312D5B4). That individual asserted they had not been aware of the article, noted inconsistencies with the data presented, did not endorse the research results reported, and stated they had consulted the other named authors, who collectively agreed the article should be retracted. The journal subsequently attempted to contact the other named authors directly, but none of them were responsive. The journal is ultimately retracting this article, because the editor no longer believes the data presented in Figure 2 provides confidence in the construction of the liv4Δ mutant, which anchors the study.The last-named author contacted the journal editor in May 2023, after being alerted to concerns with duplicated images in Figure 2, which had been posted on PubPeer in November 2021 (


E. coli, and yeast. Plants may be under an even heavier DNA damage threat from abiotic stress, reactive oxygen species leaking from the photosynthetic system, and reactive secondary metabolites. In general, BER in plant species is similar to that in humans and model organisms, but several important details are specific to plants. Here, we review the current state of knowledge about BER in plants, with special attention paid to its unique features, such as the existence of active epigenetic demethylation based on the BER machinery, the unexplained diversity of alkylation damage repair enzymes, and the differences in the processing of abasic sites that appear either spontaneously or are generated as BER intermediates. Understanding the biochemistry of plant DNA repair, especially in species other than the Arabidopsis model, is important for future efforts to develop new crop varieties.Base excision DNA repair (BER) is a key pathway safeguarding the genome of all living organisms from damage caused by both intrinsic and environmental factors. Most present knowledge about BER comes from studies of human cells,  DNA repair is a vital process in all living organisms . Repair E. coli and yeast systems. If base damage is present in the genome, it is recognized and excised by DNA glycosylases, lesion-specific enzymes that hydrolyze the N-glycosidic bond in nucleotides. Eleven DNA glycosylases with different substrate specificities are presently known in humans, and eight in E. coli  in DNA  are dealt with by a system known as base excision repair (BER) ,7,8. The E. coli , while s) in DNA . Others,) in DNA . All DNA) in DNA . The res) in DNA . A notabE. coli, DNA polymerase I is the major BER polymerase, and its 5′→3′ exonuclease activity degrades the downstream fragment. Finally, DNA ligase  seals the last nick restoring the integrity of DNA  in animal cells, chloroplasts generate an abundant additional ROS flow in plants ,10. LastAbiotic stress plays a critical role in plant life and is one of the main factors limiting crop productivity. Different types of abiotic stress can have a damaging effect on the genome of plant cells, both directly and through the production of reactive secondary metabolites and signaling molecules . BER actE. coli, G/T mismatch-specific thymine DNA glycosylase (TDG), single-strand-selective monofunctional uracil-DNA glycosylase 1 (SMUG1), and methyl-CpG binding domain 4-containing DNA glycosylase (MBD4) in humans. Of these, Ung/UNG and SMUG1 are general-purpose repair glycosylases removing U from any DNA context, MBD4 is specialized for U repair in damaged CpG dinucleotides, and TDG mainly participates in active demethylation rather than in DNA repair [Uracil–DNA glycosylase  was the first DNA glycosylase discovered and studied with respect to its mechanism ,32. SeveA repair ,34. WithA repair .E. coli and human UNG [A. thaliana cell extracts support the full BER cycle for U with base excision, AP site processing, dNMP incorporation, and nick ligation [Chlamydomonas reinhardtii [The first plant protein with the uracil–DNA glycosylase  activity was purified in 1983 from wheat germ and showed properties fairly typical for uman UNG . Enzymatuman UNG ,38,39,40ligation ,42. The nhardtii .Arabidopsis since the UNG gene knockout totally eliminates the activity in cell extracts and enhances resistance to 5-fluorouracil, a chemical that promotes DNA instability via excessive U accumulation and excision [ung plants do not accumulate the expected C→T mutations, suggesting the presence of alternative repair mechanisms [Cloned recombinant AtUNG removes U from both single- and double-stranded DNA, with a moderate preference for U:G pairs . AtUNG sexcision . Paradoxchanisms .Genome sequencing reveals no orthologs of human TDG and SMUG1 in flowering plants, although a TDG homolog is found in green algae and a SMUG1 homolog is present in mosses, ferns, horsetails, and gymnosperms. At the sequence level, they are more closely related to bacterial rather than to metazoan homologs, indicating possible horizontal transfer events. None of these homologs are functionally characterized.MBD4 in vertebrates, first discovered as a protein containing an MeCP2-like mC-binding domain, is thought to counteract deamination specifically in CpG dinucleotides ,46,47. TE. coli and humans, respectively) that sanitizes the dNTP pool and prevent 8-oxoG incorporation by DNA polymerases, and two DNA glycosylases, Fpg/OGG1 and MutY/MUTYH  a chance to incorporate C opposite to 8-oxoG and hand the baton to Fpg/OGG1 for the second round of repair  structural superfamily and are either monofunctional (MutY/MUTYH) or catalyze β-elimination (OGG1 and Nth/NTHL1), whereas Fpg, Nei, and NEIL1–NEIL3 come from the helix–two-turn–helix (H2TH) structural superfamily and catalyze β,δ-elimination [Oxidative DNA base damage presents the largest group of chemically diverse lesions, unavoidable in living organisms with an oxygen-based metabolism ,52. MosttY/MUTYH . Fpg andiewed in ,54). In mination ,56.A. thaliana cDNA library based on sequence homology and termed MMH-1  [fpg E. coli strain [MMH-1 and OGG1 knockout cell extracts are affected to about an equal degree [fpg phenotype when expressed in E. coli, raising questions about the substrate specificity of this protein variant [Plants possess a collection of all glycosylases for oxidative damage repair. A homolog of bacterial Fpg was cloned from an for Fpg) . The prifor Fpg) ,58,59. Tfor Fpg) , and boti strain . Interesi strain ; howeverl degree . Furtherl degree . The strl degree . Notably variant .A. thaliana, MMH homologs were partly characterized in sugarcane [Medicago truncatula) [fpg mutY E. coli reporter strain [M. sativa), MMH is upregulated in response to cell exposure to quantum dots, which apparently also cause oxidative stress [Besides ugarcane ,65 and bncatula) . The sugr strain . In a ree stress .fpg mutY-deficient E. coli [A. thaliana orthologs [fpg or fpg mutY-deficient E. coli [AtOGG1 gene is universally expressed and is induced neither by oxidative stress nor increased 8-oxodGTP caused by a deficiency of AtNUDX1, the only 8-oxodGTPase in A. thaliana [After a long history of failures to find eukaryotic orthologs of bacterial Fpg in a pre-genomic era, in 1996, a non-homologous 8-oxoguanine glycosylase, OGG1, was cloned from yeast based on the ability to complement the mutator phenotype of  E. coli  or cross E. coli . The hum E. coli ,71,72 anrthologs ,74 follo E. coli ,73,74,75thaliana . Interesthaliana , only onA. thaliana plants overexpressing OGG1 show increased seed longevity and resistance to osmotic and heat stress [A. thaliana, barrel clover, and eggplant [Transgenic t stress . OGG1 iseggplant ,66,78,79A. thaliana, but some residual activity is still observed in the double knockouts [Together, MMH-1 and OGG1 account for the majority of 8-oxoG repair in nockouts . Its natA. thaliana possesses two Nth homologs, NTH1 and NTH2 [As for oxidized pyrimidine repair, and NTH2 ,81, whicmC), serving as a reversible mark that plays an important role in transcription regulation and genome protection from mobile elements [mC residues as epigenetic marks, they are set on by DNA (cytosine-5)-methyltransferases that either methylate DNA de novo or reinstall mC in hemimethylated DNA. However, the opposite-direction process, active demethylation, employs entirely different mechanisms in animals and plants, albeit both based on BER. In animals, mC is oxidized by TET family dioxygenases first to 5-hydroxymethylcytosine, an epigenetic mark itself, and further to 5-formylcytosine and 5-carboxycytosine; all these derivatives can also be actively deaminated to 5-hydroxymethyluracil, 5-formyluracil, and 5-carboxyuracil, respectively. The oxidized/deaminated bases are excised by TDG and replaced with C through the BER pathway [mC. In 2002, two paralogous genes involved in the epigenetic control in A. thaliana were found to encode DNA glycosylases: DEMETER (DME), a gene responsible for maternal allele derepression of MEDEA Polycomb group chromatin remodeler during embryo and endosperm development, and REPRESSOR OF SILENCING 1 , an activator of transcription of several endogenous genes acting throughout the life cycle [DEMETER-LIKE 2 (DML2) and DEMETER-LIKE 3 (DML3), were characterized shortly thereafter [Gene expression in higher eukaryotes is controlled through several mechanisms, of which epigenetic DNA methylation is especially prominent. In most animals and plants, it relies on a “fifth nucleobase”, 5-methylcytosine  domain ,109. ThemC and T, it efficiently removes 5-hydroxyuracil and 5-hydroxymethylcytosine, has moderate activity on 5-fluorouracil, 5-bromocytosine, and 5-bromouracil, and possesses only marginal ability to excise 5-hydroxymethyluracil [mC excision, which again may be important to prevent mutagenesis by misincorporation during BER [mC:G pairs using a trio of residues  that intercalate into the DNA helix [mC removal in vitro and male fertility in vivo, despite being missing in all other HhH glycosylases [mC reader targeting the complex to methylated genome regions [Of all DML proteins, ROS1 is probably the best investigated in terms of its enzymatic properties. In addition to yluracil ,107,112.yluracil ,114,115.yluracil . With reyluracil ,107. Damring BER . The enzNA helix . The iroNA helix ,121,122.osylases ,123. Likosylases . The seaosylases ,124,125.osylases . In vivoosylases . ROS1 is regions .Despite the presence of RRM domains, it is not clear whether DML proteins are guided to their site of action by any RNA. In vivo, the proper targeting of ROS1 depends on ROS3, a small RNA-binding protein, but physical interaction between ROS1 and ROS3 has not been demonstrated . DemethyThe biochemistry of DML2 and DML3 has received much less attention, but both enzymes seem to be similar to DME and ROS1 in terms of the substrate specificity and reaction mechanism, although DML2 lacks thymine-excising activity ,89. It rmC marks are laid out, plants experience more local methylation reconfiguration in the early zygote [MEA, FLOWERING WAGENINGEN (FWA), FERTILIZATION INDEPENDENT SEED 2 (FIS2), or FLOWERING LOCUS C (FLC) in A. thaliana [DME, so far, seems to act only in the regulation of plant development. Unlike mammals, in which a global wave of demethylation occurs immediately after fertilization and then new y zygote ,135. DMEthaliana ,138,139.thaliana . Both DMthaliana . In vegethaliana , which ithaliana .Boea hygrometrica) [Other DML proteins are also partially involved in development regulation. For example, in wheat (eleven homologs), barley (three homologs), and rice (six homologs), “DME” and “ROS1a” derepress genes coding for prolamins, the main storage proteins, during seed development ,102. In metrica) .ros1 A. thaliana plants show a dramatic increase in methylation of some endogenous promoters, such as RD29A, FWA, or EPF2, while overexpression of ROS1 induces their demethylation and activation [GP1, LINE1-4, SN1, MU1) in plant cells [Tos17 retrotransposon, a Copia family element widely used for experimental insertion mutagenesis in rice [A. thaliana requires ROS1, DML2, and DML3 to mount an immune response to Pseudomonas syringae infection [ROS1, DML2, and DML3 primarily regulate gene expression in somatic tissues. Mutant tivation ,146,147.nt cells . One of  in rice . Rice DM in rice  and are  in rice . The tom in rice ,153. The in rice . A. thalnfection , while infection .ros1 dml2 dml3 mutants [dme ros1 dml2 dml3 mutants lacking all DML proteins in somatic tissues can be obtained by central cell-specific ectopic expression of DME and display global hypermethylation and loss of tissue-specific differential methylation [Despite an apparently wider range of processes and targets in comparison with DME, the methylation control by ROS1 seems to be limited to a few loci. Although ROS1/DML2/DML3-targeted regions contain many methylation-directing siRNA targets, at least ROS1 seems to remove methyl labels whose origin is not limited to RNAi-dependent pathways ,156. Str mutants . Bisulfi mutants ,139. QuaROS1 itself is positively controlled by methylation: a helitron transposon is present in front of the ROS1 promoter and suppresses transcription, whereas a specific “methylation monitoring sequence” in between can be methylated to uncouple the helitron from the promoter and boost ROS1 expression [DML genes are also regulated by miRNA in development and during abiotic stress, as documented in A. thaliana, barley, and red sage  [Interestingly, expression of pression ,158. DMLorrhiza) ,159,160.Although the full spectrum of plant cell responses involving DML glycosylases has not yet been elucidated, it is clear that these proteins orchestrate gene activity in development and integrate many responses to various kinds of abiotic stress. In dicots, DME seems to be strictly dedicated to developmental regulation, while other DMLs participate both in development and in the stress response. In monocots, such specialization is not yet clear. A concept on “stress memory” has been proposed, in which the methylation status changes in response to abiotic stress can be imprinted and stably maintained for some time in plant development , or evenmC epigenetic label [On a more practical side, the expression of transgenes in plants is often regulated by methylation-sensitive elements, such as cauliflower mosaic virus 35S promoter, and regulation of ROS1 was proposed as a way to drive or silence the expression of transgenes . DML glyic label ,164,165,ic label ,167. HowN3 and N7 positions, whereas products of alkylation at exocyclic groups and other ring nitrogens  are repaired by Ada-like alkyltransferases and AlkB-like dioxygenases without base excision [E. coli, the AlkA protein, which belongs to a wide, functionally diverse HhH superfamily, is induced by alkylation stress, and removes N3- and N7-alkylpurines and 1,N6-ethenoadenine (εA). Another E. coli alkylpurine–DNA glycosylase, Tag, is constitutive, and its substrate specificity is limited to N3-alkyladenine; structurally, it is sometimes also grouped with HhH proteins, but outside of the namesake helix–hairpin–helix motif, it is very different from the rest of the superfamily. In human cells, MPG protein  removes 3-methylpurines, hypoxanthine, and εA and belongs to a completely different structural family, resembling the C-terminal domain of methionyl-tRNAfMet formyltransferase [E. coli) possess DNA glycosylases belonging to the HEAT repeat domain and winged-helix domain superfamilies and specific for alkylated purines or purine adducts with some natural genotoxic antibiotics [The major products of electrophilic addition removed by BER are purines alkylated at excision ,169,170.nsferase ,172. Moribiotics ,174.S-adenosyl methionine [In vertebrates, the primary endogenous source of DNA alkylation is stray reactions with metabolic methyl group donors, mainly thionine . Many plthionine ,14. Thusthionine  or mismathionine , and rinthionine , yet BERA. thaliana genome [A. thaliana possesses seven TAG homologs, some of which appear to result from ancestral duplications, while others appeared recently  structural superfamily) and endonuclease IV  [All three types of DNA glycosylase products, namely AP site, 3′-terminal unsaturated aldehyde, and 3′-terminal phosphate, need further processing to expose a 3′-OH end that can be extended by DNA polymerases . In E. crfamily) . Xth is rfamily) ,187. APErfamily) .A. thaliana enzymes belonging to the EEP superfamily, AtAPE1L, AtARP, and AtAPE2, show an AP endonuclease activity [ape1L ape2 and ape1L zdp double knockout seeds are inviable, whereas those of the arp ape1L and arp ape2 genotype survive [Three activity ,190. Addactivity . Of the  survive ,193. APEape1L and ape1L zdp knockout cells [APE1L interacts with ROS1 forming a ternary complex on DNA, but does not displace ROS1 from the reaction product . This diut cells .A. thaliana, wheat, and sugarcane demonstrate the AP endonuclease activity [A. thaliana arp plants show sensitivity to methyl methanesulfonate and tert-butyl hydroperoxide, but not to H2O2 [ARP proteins from activity ,196,197 activity  and to pactivity ,198. A. In addition to their AP endonuclease activity, mammalian APE1 proteins possess so-called “redox activity”, reactivating many oxidized transcription factors . ARP haszdp ape2 double knockouts, 3′-blocked BER intermediates accumulate and cause proliferation and development defects partially rescued by abrogation of MMH glycosylase/β,δ-lyase or of SOG1, a plant-specific transcriptional regulator acting in a break-sensing ATR pathway [The APE-less BER branch in plants relies on the zinc finger DNA 3′-phosphatase (ZDP), rather than on PNKP as in mammals . AtZDP e pathway . Thus, AA. thaliana was shown to depend on the sequential action of MMH-1 and ZDP [N7-methylguanine, which either depurinates spontaneously or is excised by the strictly monofunctional alkylpurine glycosylases, producing an AP site. It has been suggested that, at least in plants, ARP and/or APE1L may be predominantly involved in the repair of AP sites formed by DNA glycosylases, whereas AP lyases contribute significantly to the repair of spontaneously formed AP sites [DNA glycosylases/AP lyases can also process AP sites, formed either by spontaneous depurination or by other DNA glycosylases. Until recently, this reaction was considered rather irrelevant biologically because the processing of the 3′-ends would still require AP endonucleases or 3′-phosphatases. However, recently the repair of damage induced by methyl methanesulfonate (MMS) in  and ZDP . MMS preAP sites ,207.A. thaliana cell extracts are proficient in both short- and long-patch BER [atch BER ,195,208.atch BER ,209,210.atch BER ,212. As atch BER ,213. Moratch BER , AtPOLλ atch BER . All thiA. thaliana POLγ homologs are often referred to as homologs of E. coli DNA polymerase I (PolI), albeit they are actually closer to POLγ. Mutations in the small subunit of POLδ, POLD2, are hypersensitive to MMS, which may suggest POLδ involvement in BER [There definitely is a possibility that other plant DNA polymerases, whose main role is in replication, translesion synthesis, or other DNA repair pathways, also participate in BER. In addition to POLλ, plant genomes code for homologs of human DNA polymerases γ and θ (Family A), α, δ, ε, ζ (Family B), η, κ, and REV1 (Family Y). In the literature, for historical reasons, two t in BER . The POLA. thaliana FEN1 was independently cloned by two groups in 2016, as a suppressor of transgene silencing and a regulator of light-dependent root and hypocotyl elongation [rad27 yeast strain deficient in FEN1 [Flap endonuclease, FEN1, is a key enzyme in long-patch BER in vertebrates, removing the strand displaced by a DNA polymerase once it progresses beyond the replacement of a single nucleotide ,218. FENongation ,220. Bioongation ,220. FENongation ,223,224.A. thaliana) seems to be the only DNA ligase acting in plant BER [A. thaliana also possesses a second homolog of Lig I (At1g49250), which remains uncharacterized at the moment.In vertebrates, two DNA ligases participate in different BER branches: Lig IIIα completes short-patch BER, and Lig I works in the long-patch subpathway . As withlant BER ,225. Genlant BER ,226,227.A. thaliana [A. thaliana plants deficient in XRCC1 are viable but radiosensitive [XRCC1, the adaptor protein, orchestrates the coordinated transfer of repair intermediates between sequential BER enzymes in higher eukaryotes ,229. Plathaliana  and ricethaliana . Notablythaliana ,233. Nevthaliana . Co-locathaliana  may be densitive .A. thaliana possesses only three PARPs; in the literature, two of them are somewhat non-discriminately named either PARP1 or PARP2. AtPARP1 (At2g31320) seems to be the primary poly(ADP-ribose) polymerase in DNA damage response, while AtPARP2 (At4g02390) is more active as a recombinant protein in vitro, but apparently plays a back-up role when assayed in cell-free extracts [Poly(ADP-ribose) polymerases are multifunctional proteins sensing single- and double-strand breaks and initiating post-translational modification of many proteins, including themselves by mono- and poly(ADP-ribose) ,236. In extracts ,238. AtPRP2 At4g00 is moreRP2 At4g00 is moreextracts . OverallBER in organelles is organized along with the general BER scheme, but has some important details. The best-studied example of organellar BER is provided by mammalian mitochondria ,242. TheA. thaliana and potato tuber mitochondria can support the full BER cycle for U [UNG gene, which produces mitochondrial UNG1 and nuclear UNG2 isoforms with different N-terminal tails via alternative transcription initiation and splicing, only one AtUNG mRNA and protein exist; however, the first 174 residues of AtUNG have been shown to target an EGFP reporter to mitochondria [ung A. thaliana plants do not accumulate mutations in mitochondrial DNA, and the same holds for mmh and ogg1 plants, suggesting the presence of alternative repair mechanisms, most likely mismatch repair [Araucaria angustifolia) contain activities of UNG, MMH, and AP endonuclease [A. thaliana, rice, and tobacco [le for U ,244, andchondria . Intereschondria . This ischondria ,246. An chondria , is the h repair . UNG acth repair , and mitnuclease . Pol I/P tobacco ,253,254, tobacco , one can tobacco . The mitA. thaliana, Lig I is targeted to nuclei and mitochondria, but not to chloroplasts [In comparison, BER in chloroplasts has received less attention. Regarding the initial BER stages, UNG activity was detected in maize chloroplasts  and AtNTroplasts ,259. TheAlthough the principal mechanism of base excision DNA repair in plants is the same as in vertebrates, bacteria, and yeast, the evolutionary history and specific lifestyle endowed plants with their unique repair features. The most obvious and seemingly the most important of those is the existence of epigenetic DNA glycosylases, subverting the genome protection machinery for the ends of gene activity regulation. No equivalent of this system is known outside of the Viridiplantae kingdom; the animal TETs/TDG active demethylation pathway apparently evolved independently. Another striking feature of plant BER, still virtually unstudied, is the high diversity of DNA glycosylases homologous to proteins repairing alkylation damage in bacteria. Elucidating their specificity and biological roles might shed light on the unique kinds of genotoxic stress to which plants are exposed as compared to other living things. Processing of DNA glycosylase reaction products, namely AP sites and dirty 3′-ends, also seems to vary between plants on the one hand and humans and bacteria on the other hand, and relies more on AP lyases and 3′-phosphatases in plants, although there is still no clear picture of distinct subpathways following the action of different glycosylases. Finally, chloroplasts, the most distinctive plant organelle, are an exceptionally important source of DNA damage, yet the repair of the chloroplast genome has been barely addressed so far. Understanding the biochemistry of plant DNA repair, especially in the key crop species, such as rice, wheat, vegetable cultures, etc., should advance our ability to breed or engineer new varieties with increased robustness to abiotic stress.