ETS-driven prostate cancer initiation and progression remain poorly understood due to a lack of model systems that recapitulate this phenotype. We generated a genetically engineered mouse with prostate-specific expression of the ETS factor, ETV4, at lower and higher protein dosage through mutation of its degron. Lower-level expression of ETV4 caused mild luminal cell expansion without histologic abnormalities, and higher-level expression of stabilized ETV4 caused prostatic intraepithelial neoplasia (mPIN) with 100% penetrance within 1 week. Tumor progression was limited by p53-mediated senescence and Trp53 deletion cooperated with stabilized ETV4. The neoplastic cells expressed differentiation markers such as Nkx3.1 recapitulating luminal gene expression features of untreated human prostate cancer. Single-cell and bulk RNA sequencing showed that stabilized ETV4 induced a previously unidentified luminal-derived expression cluster with signatures of cell cycle, senescence, and epithelial-to-mesenchymal transition. These data suggest that ETS overexpression alone, at sufficient dosage, can initiate prostate neoplasia.The mechanisms underlying  Sufficient protein dosage of ETV4 can directly transform prostate epithelial cells, which recapitulates early human disease. ETS family transcription factors including ERG and three highly conserved polyomavirus enhancer activator 3 (PEA3) subfamily members—ETV1, ETV4, and ETV5—are prevalent and mutually exclusive in prostate cancer; they occur early in the natural history of the disease  models that overexpress ERG or ETV1 showed either minimal or no phenotype early time points  and stabilized ETV4 , harboring mutations at the COP1-binding sites and therefore resistant to COP1-mediated degradation. We demonstrate that the stabilized ETV4AAA significantly increased the protein dosage of ETV4. ETV4AAA expression alone initiates widespread mPIN throughout the prostate epithelium within 2 weeks of activation. The mPIN fails to further progress to invasive cancer due to ETV4-mediated simultaneous activation of the p53-dependent senescence program, and ETV4AAA cooperates with Trp53 loss to promote the development of focally invasive prostate cancer. The neoplastic cells retain expression of prostate lineage differentiation markers such as Nkx3.1 similar to most cases of localized human prostate cancer. In contrast, Pten loss–driven murine prostate cancers generally lose Nkx3.1 and are de novo castration resistant and distinct in human primary adenocarcinoma . We expressed enhanced green fluorescent protein (EGFP) vector control, ETV4WT, and ETV4AAA in A375 melanoma cells and found that, at baseline, protein level of ETV4AAA was significantly higher than that of ETV4WT. Treatment with MG132 that inhibits proteosomal degradation increased ETV4WT protein levels and endogenous ETV4 in vector-infected control cells but only minimally affected ETV4AAA protein level, indicating that ETV4AAA is constitutively stable (fig. S1A). To evaluate the possibility that, beyond protein stability, the mutations introduced in ETV4AAA may alter its transcriptional function, we expressed EGFP, ETV4WT, and ETV4AAA in two prostate cancer cell lines, 22Rv1 and PC3, and performed RNA sequencing (RNA-seq) analysis. In 22Rv1 cells, the protein level of ETV4AAA was significantly higher than that of ETV4WT due to degradation by COP1. PC3 cells harbor a deletion in COP1 and have increased baseline protein levels of ETV4 (AAA and ETV4WT exhibited similar protein levels (fig. S1B). Principal components analysis (PCA) of RNA-seq showed that the first principal component (PC) was separated by cell line and that the second PC was separated by ETV4 expression. In 22RV1 cells, ETV4AAA caused a much greater expression change as ETV4WT in the same direction. In PC3 cells that have baseline higher levels of ETV4, the transcriptome perturbation was less in general, and ETV4WT caused a greater change in gene expression (fig. S1C). We compared the global transcriptome change induced by ETV4AAA and ETV4WT. In 22Rv1 cells, there was a high correlation [Pearson correlation coefficient (r) = 0.72], and ETV4AAA induced a greater change (fig. S1D). In PC3 cells, the global transcriptome change was also strongly correlated (Pearson r = 0.76), and ETV4WT induced slightly higher gene expression changes. These data suggest that ETV4AAA regulates a similar transcriptome to ETV4WT.Previous studies showed that transgenic and conditional WTETV4 and AAAETV4 followed by IRES-EGFP driven by the CAG promoter knocked into the Rosa26 locus  mice  with enlarged nuclei, prominent nucleoli, and cribriform growth in the anterior and dorsal prostate (WTETV4-expressing mice were indistinguishable from those expressing EYFP. While GFP immunohistochemistry (IHC) showed prevalent EGFP- or EYFP-positive cells in murine prostates of all three genotypes, ETV4 IHC detected weak nuclear ETV4 protein in ETV4WT prostate but strong nuclear ETV4 expression in the AAAETV4-expressing prostate cells . This highlights a lobe-specific sensitivity to specific oncogenic transformation as observed in other genetic engineered models of prostate cancer .To determine whether high ETV4 protein expression alone is sufficient to drive progression to invasive cancer, we administered TAM in 6- to 8-week-old mice and aged them for 6, 9, and 12 months for phenotypic assessment. Unexpectedly, the mPIN of the anterior and dorsal prostate of  ETV4AAA . The proAAA in the mouse anterior prostate is comparable to that in human prostate cancer, we performed ETV4 IHC using the same conditions on a patient-derived xenograft (PDX) model with TMPRSS2-ETV4 fusion (MDA PCa 175-6) and a control PDX (MDA PCa 173-2) from MD Anderson (MDA PCa program) deposited in biobank , the most commonly used Cre driver in the prostate epithelium, that mediates continued recombination in prostate epithelial cells from puberty . The areas of mPIN were marked by positive EGFP staining. We reasoned that the continuous induction of ETV4AAA though a nonconditional Cre driver results in relatively constant phenotype over time. Despite the continuous induction that maintained mPIN throughout the time studied, there was no evidence of disease progression over time. These data suggest that the expression of AAAETV4 can directly induce mPIN with little latency and that persistent mPIN phenotype requires maintenance of the AAAETV4 expression in prostate epithelial cells; however, AAAETV4 expression alone is insufficient to mediate progression to invasive cancer.We chose ETS translocation alters the chromatin enhancer landscape and the AR cistrome in prostate cancer  and RNA-seq, respectively. We chose the 2-week time point to enrich for direct targets. To specifically analyze prostate epithelial cells with the recombined allele, we sorted EPCAM-positive and EYFP- or EGFP-positive prostate epithelial cells by fluorescence-activated cell sorting (FACS) from T2; EYFP, WTT2; ETV4, and AAAT2; ETV4 murine prostates (fig. S4A).We next examined the molecular impact of stabilized ETV4 in the unique phenotypes associated with prostate tumorigenesis. Recent data have shown that WTETV4- and EYFP-expressing cells clustered closely, while AAAETV4-expressing cells exhibited marked changes in the accessible chromatin landscape  > 2], with approximately equal number of peaks with significantly increased and decreased ATAC signals . The FOX and AP1 motifs were significantly enriched at both ETV4AAA–up-regulated and ETV4AAA–down-regulated peaks, and the nuclear receptor motif was enriched at the down-regulated peaks . Unsupervised clustering of ATAC signal intensity showed that andscape . We foun signals . Among tagnitude , indicated peaks . Consistpression  .eq sites . ATAC-seAAA-induced transcriptome changes. Compared to EYFP-expressing control, AAAETV4 expression induced a greater number of statistically up-regulated genes (537) than down-regulated genes (250)   . Hierarc control . Integra control . These dAAA in prostate epithelial cells, we performed gene set enrichment analysis (GSEA) using ~3000 curated genes sets and custom prostate cancer gene sets. There were many more gene sets enriched among genes significantly up-regulated by ETV4AAA expression than among genes down-regulated by ETV4AAA expression, also suggesting that ETV4AAA is primarily a transcriptional activator (fig. S4D). Among enriched gene sets are those genes associated with cell cycle and proliferation (CHANG_CYCLING_GENES), prostate cancer tumorigenesis in human prostate cancer (TCGA_PrCa_UP), and ERG target genes in prostate cancer (VCAP_siERG_DN and VCAP_Lenti_ERG_UP)  . We furtWT- and ETV4AAA-expressing prostate cells; canonical ETS transcriptional targets, e.g., Dusp6 and Plat, were mildly induced by ETV4WT and much more significantly induced by ETV4AAA (Trp53), its downstream target p21 (Cdkn1a), and senescence-associated genes Cdkn2b, Cxcl1, Spp1, and Tgfb1 were all significantly up-regulated in ETV4AAA-expressing prostate cells compared to that in EYFP and ETV4WT controls. ETV4AAA prostates stained positive for p53 by IHC and for senescence-associated β-galactosidase –EGFP was similar between ETV4 ETV4AAA . p53  is significantly higher in ETS fusion–positive samples (42.42%) than that in ETS fusion–negative samples (15.83%), consistent with analysis of the Memorial Sloan Kettering Cancer Center cohort . These data indicate that aberrant high level of ETS expression activates p53 and downstream senescence programs and that p53 loss might be a cooperative event that overcomes the barrier for human prostate tumorigenesis and progression.We analyzed the TCGA dataset of primary prostate cancer to correlate r cohort  (20). Wee cancer . To assee cancer : (i) TCGAAA-mediated senescence and tumor progression, we crossed in the LoxP/LoxPTrp53 mice into the AAAT2; ETV4 mice to generate LoxP/LoxP; ETV4AAAT2; Trp53 mice. Two weeks after TAM injection, Western blot of prostate lysates shows decreased p53 protein , which include 19,854 genes, with a median of 1973 genes per cell and a median of 2039 cells per mouse. To visualize single cells of the global atlas, we used uniform manifold approximation and projection (UMAP) for dimension reduction. We then performed Leiden clustering  on FACS-sorted live cells from prostates of T2; EYFP wild-type mice, there was expansion of epithelial cells  over time from 2 weeks to 4 months  , L2, and L3 that had been found in previous studies (fig. S5A and table S6)  that highly expresses lateral prostate marker genes Msmb and Trpv6 (Tacstd2 (Trop2), Ly6a (Sca1), Psca, and Krt4; and exhibit enhanced progenitor characteristics (Foxi1 (fig. S5A and table S6).We next analyzed the identities of luminal cell clusters by the marker gene expression. We then annotated the luminal clusters based on the highly expressed marker genes and quantified the cell numbers in each group (fig. S5A). We first focused on T2; EYFP wild-type prostates, WTT2;ETV4 prostates did not comprise any new luminal clusters, suggesting that ETV4WT cannot transform luminal cells. However, WTT2;ETV4 prostates exhibited expansion of Luminal_1 clusters compared with T2;EYFP that is more pronounced from 2 weeks to 4 months . Furthermore, within the WTT2;ETV4 prostates, the expansion of Luminal_1 clusters over time was mostly due to WTETV4-expressing cells (dashed green and dashed cyan). These data suggest that, while expression of WTETV4 did not cause detectable histologic phenotype or new gene expression cluster, its expression was able to cause luminal expansion over time at the single-cell level.Compared to AAA in AAAT2; ETV4 and AAA;Trp53LoxP/LoxPT2; ETV4 prostates generated two new luminal clusters that express AAAETV4  with distinct transcriptome features from previously described luminal clusters  cluster  using GSEA using GSEAPY package . Pathway analysis showed that extracellular matrix (ECM) organization and multiple growth factor pathways are up-regulated (fig. S6B). To verify the scRNA-seq results, we analyzed the gene expression of L2 marker gene Tacstd2/Trop2 using immunofluorescence staining (fig. S6C). While EGFP (indicating ETV4)–positive cells are Tacstd2 negative, there is a marked expansion of Tacstd2-positive cells in AAAETV4 mice and LoxP/LoxP; ETV4AAATrp53 mice. These Tacstd2-positive cells were found in regions of EGFP-positive, ETV4AAA-expressing cells, suggesting that ETV4AAA-positive cells, through paracrine interactions, expand the normal L2 population.In addition to ETV4 ETV4AAA . This suT2; EYFP and WTT2; ETV4 mice, Basal_2 cells were found exclusively in ETV4AAA-expressing mice, and Basal_3 cells had an intermediate gene expression and were found in all groups of mice (fig. S7A). Basal_2 cells highly express genes Col17a1, Areg, Ly6d, and Cd44 (fig. S7B). We performed immunofluorescence staining of Col17a1 and found a high expression in Ck5-positive basal cells adjacent to EGFP-positive (ETV4AAA-expressing) luminal cells in ETV4AAA mice (fig. S6D), suggesting that ETV4AAA expression in the luminal cells induced paracrine gene expression changes in basal cells.We next identified and quantified subclusters in basal, stroma, and hemopoietic populations. We identified four basal expression clusters. Basal_1 cells were found predominantly in Lama2, Zeb1, Wnt2, Wnt6, Wnt10a, and Rorb; Mesenchymal_2 is characterized by expression of Sult1e1, Fgf10, and Rspo1; Myofibroblast is characterized by expression of Acta2, Myh11, and Notch3. These clusters represent previously described Mesenchymal_1, Mesenchymal_2, and myofibroblast subsets (table S6) . Mesenchymal_3 is characterized by expression of Sod3 and Osr1, which are mesenchymal stem cell markers .We identified five stroma clusters: Mesenchymal_1 to Mesenchymal_4 and myofibroblast (fig. S7C). Mesenchymal_1 is characterized by expression of The myeloid cells and lymphocytes are characterized using the expression of the marker genes (fig. S7F). Marker genes of M1 macrophages, M2 macrophages, and myeloid-derived suppressor cells (MDSCs) are adopted from other publications  level and a high dosage (ETV4AAA) level of ETV4 protein through increased ETV4 protein stability by mutating the conserved ExxVPD motifs responsible for COP1 degradation  is significantly higher in ETS fusion–positive samples than that in ETS fusion–negative samples . MecAAA-driven murine prostate cancer model is that it maintains the characteristics of early-stage human prostate cancers, including prostate differentiation markers, such as Nkx3.1 expression. Comparably, prostates of Pten deletion, the most well-studied GEM model of prostate cancer, exhibit increased basal marker expression, lose Nkx3.1 expression, and are castration resistant de novo  and mutated ETV4AAA cDNA into the plasmid in the middle of the two cassettes. The targeting plasmid was electrophoresed into albino C57BL/6J embryonic stem cells, and G418-resistant clones were isolated by the standard procedures. The clones were screened by Southern blotting. Positive clones were injected into C57BL/6J blastocysts by the MSKCC Mouse Genetics Core Facility, and chimeras were mated with albino C57BL/6J females. Germline transmission was confirmed in albino offspring using Southern blotting. For subsequent generations, genotyping was performed by quantitative polymerase chain reaction (PCR) using the following primers: hETV4_E3_F: GCCGCCCCTCGACTCTGAA; hETV4_E4_R: GAGCCACGTCTCCTGGAAGTGACT.Rosa26 targeting was as described by Srinivas GTACCAGAC(GCAGCAGCC)AGTGATGAGCAGTTTGTTCCTGAT(GCTGCTGCT)TTCCATTCAGAAAACCTAGCTTTCCACAGCCCCACCACCAGGATCAAGAAGGAGCCCCAGAGTCCCCGCACAGACCCGGCCCTGTCCTGCAGCAGGAAGCCGCCACTCCCCTACCACCATGGCGAGCAGTGCCTTTACTCCAGTGCCTATGACCCCCCCAGACAAATCGCCATCAAGTCCCCTGCCCCTGGTGCCCTTGGACAGTCGCCCCTACAGCCCTTTCCCCGGGCAGAGCAACGGAATTTCCTGAGATCCTCTGGCACCTCCCAGCCCCACCCTGGCCATGGGTACCTCGGGGAACATAGCTCCGTCTTCCAGCAGCCCCTGGACATTTGCCACTCCTTCACATCTCAGGGAGGGGGCCGGGAACCCCTCCCAGCCCCCTACCAACACCAGCTGTCGGAGCCCTGCCCACCCTATCCCCAGCAGAGCTTTAAGCAAGAATACCATGATCCCCTGTATGAACAGGCGGGCCAGCCAGCCGTGGACCAGGGTGGGGTCAATGGGCACAGGTACCCAGGGGCGGGGGTGGTGATCAAACAGGAACAGACGGACTTCGCCTACGACTCAGATGTCACCGGGTGCGCATCAATGTACCTCCACACAGAGGGCTTCTCTGGGCCCTCTCCAGGTGACGGGGCCATGGGCTATGGCTATGAGAAACCTCTGCGACCATTCCCAGATGATGTCTGCGTTGTCCCTGAGAAATTTGAAGGAGACATCAAGCAGGAAGGGGTCGGTGCATTTCGAGAGGGGCCGCCCTACCAGCGCCGGGGTGCCCTGCAGCTGTGGCAATTTCTGGTGGCCTTGCTGGATGACCCAACAAATGCCCATTTCATTGCCTGGACGGGCCGGGGAATGGAGTTCAAGCTCATTGAGCCTGAGGAGGTCGCCAGGCTCTGGGGCATCCAGAAGAACCGGCCAGCCATGAATTACGACAAGCTGAGCCGCTCGCTCCGATACTATTATGAGAAAGGCATCATGCAGAAGGTGGCTGGTGAGCGTTACGTGTACAAGTTTGTGTGTGAGCCCGAGGCCCTCTTCTCTTTGGCCTTCCCGGACAATCAGCGTCCAGCTCTCAAGGCTGAGTTTGACCGGCCTGTCAGTGAGGAGGACACAGTCCCTTTGTCCCACTTGGATGAGAGCCCCGCCTACCTCCCAGAGCTGGCTGGCCCCGCCCAGCCATTTGGCCCCAAGGGTGGCTACTCTTACTAG.ATGGAGCGGAGGATGAAAGCCGGATACTTGGACCAGCAAGTGCCCTACACCTTCAGCAGCAAATCGCCCGGAAATGGGAGCTTGCGCGAAGCGCTGATCGGCCCGCTGGGGAAGCTCATGGACCCGGGCTCCCTGCCGCCCCTCGACTCTGAAGATCTCTTCCAGGATCTAAGTCACTTCCAGGAGACGTGGCTCGCTGAAGCTCAGVPD(AAA)SDEQFVPD(AAA)FHSENLAFHSPTTRIKKEPQSPRTDPALSCSRKPPLPYHHGEQCLYSSAYDPPRQIAIKSPAPGALGQSPLQPFPRAEQRNFLRSSGTSQPHPGHGYLGEHSSVFQQPLDICHSFTSQGGGREPLPAPYQHQLSEPCPPYPQQSFKQEYHDPLYEQAGQPAVDQGGVNGHRYPGAGVVIKQEQTDFAYDSDVTGCASMYLHTEGFSGPSPGDGAMGYGYEKPLRPFPDDVCVVPEKFEGDIKQEGVGAFREGPPYQRRGALQLWQFLVALLDDPTNAHFIAWTGRGMEFKLIEPEEVARLWGIQKNRPAMNYDKLSRSLRYYYEKGIMQKVAGERYVYKFVCEPEALFSLAFPDNQRPALKAEFDRPVSEEDTVPLSHLDESPAYLPELAGPAQPFGPKGGYSY.MERRMKAGYLDQQVPYTFSSKSPGNGSLREALIGPLGKLMDPGSLPPLDSEDLFQDLSHFQETWLAEAQPb-Cre4 Hze/JB6.Cg-Gt(ROSA)26Sor) mice of conditional CAG-driven EYFP expression were purchased from the Jackson Laboratory  in which exons 4 and 5 are flanked by LoxP sites were used as previously described . For mouse prostates, all tissues were fixed at 4°C overnight in 4% paraformaldehyde. Tissue processing, embedding, sectioning, and IHC staining was performed. Antibodies for IHC and Western blotting are as follows: chicken anti-GFP , rabbit anti-AR , rabbit anti-ETV4 , rabbit anti-Ki67 , rabbit anti-SMA , rabbit anti-p53 , rabbit anti-Nkx3.1 , mouse anti–β-actin , and mouse anti–glyceraldehyde-3-phosphate dehydrogenase . Tissue paraffin embedding, sectioning, and hematoxylin and eosin (H&E) staining were performed by the MSKCC core facility. IHC was performed by the MSKCC molecular cytology core using a Ventana Discovery XT. To generate lysates for Western blotting, tissue was homologized in radioimmunoprecipitation assay buffer using the FastPrep-24 system with Lysing Matrix A .Immunofluorescence staining of Tacstd2/Trop2 , Col17a1 , and Ck5  was performed on frozen sections. Sections were permeabilized with 0.5% Triton X-100 for 10 min and blocked with 10% normal donkey serum for 30 min at room temperature. Primary antibodies were incubated overnight. Secondary antibodies with Alexa Fluor 647 and Alexa Fluor 555 (Thermo Fisher Scientific) conjugation were applied on the second day. Image were taken with Leica TCS SP5 upright confocal microscope.Intraperitoneal injection of TAM was administered in 8-week-old mouse. Two weeks after TAM treatment, mouse prostate was digested 1 hour with collagenase/hyaluronidase  and then 30 min with TrypLE Express Enzyme  at 37°C to isolate single prostate cells. The prostate cells were stained with phycoerythrin/cyanine 7–conjugated anti-mouse CD326 (EPCAM) antibody , and then, CD326 and EYFP double-positive cells were sorted out by flow cytometry, which are luminal cells mainly from anterior prostate and dorsal prostate. The mRNA or genomic DNA was extracted from these double-positive cells and then was used for ATAC-seq and RNA-seq analysis.https://github.com/kundajelab/atac_dnase_pipelines). Briefly, paired-end reads were trimmed, filtered, and aligned against mm9 using Bowtie2  ranges between 28 and 33, suggesting that the libraries have a high quality and were able to capture the majority of regions of interests.ATAC-seq was performed as previously described (P < 0.01 and |log2(FC)| > 2. For visualization, coverage bigwig files were generated using bamCoverage command from deepTools2 and were normalized using the size factor generated by DESeq2. The differential ATAC-seq peak density plot was generated with deepTools2 ] × [−log] as input for GSEA analysis using “Run GSEA Pre-ranked” with 1000 permutations (The extracted RNA was processed for RNA-seq by the Integrated Genomics Core Facility at MSKCC. The libraries were sequenced on an Illumina HiSeq-2500 platform with 51–base pair (bp) paired-end reads to obtain a minimum yield of 40 million reads per sample. The sequenced data were aligned using STAR v2.3  calculated by DESeq2. P values were estimated using Wilcoxon rank t test and Student’s t test.To assign ATAC-seq peaks to genes, ChIPseeker_v1.12.1 . After euthanasia, the prostates were dissected out and minced with scalpel and then processed for 1 hour of digestion with collagenase/hyaluronidase  and 30 min of digestion with TrypLE . Live single prostate cells were sorted out by flow cytometry as DAPI−. For each mouse, 5000 cells were directly processed with 10x Genomics Chromium Single Cell 3′ GEM, Library & Gel Bead Kit v3, according to the manufacturer’s specifications. For each sample, 200 million reads were acquired on NovaSeq platform S4 flow cell.2(X + 1)–transformed for analysis of the combined dataset. The top 1000 highly variable genes were found using SCANPY (version 1.6.1) (Reads obtained from the 10x Genomics scRNAseq platform were mapped to mouse genome (mm9) using the Cell Ranger package (10x Genomics). True cells are distinguished from empty droplets using scCB2 package (https://arxiv.org/abs/1802.03426). We then performed Leiden clustering  package  .


Euphorbiatithymaloides is one such medicinal plant that hasgained importance but is often confused with other plants of the samespecies. In order to address this issue, this study aimed to conducta conventional and molecular pharmacognostic study for the identificationof the root of E. tithymaloides. Theroot of the plant was studied for the macroscopic observations, andthen, the root was ground into coarse powder for microscopic studiesand to determine the physiochemical properties. The powder was subjectedto extraction with solvents such as ethanol, ethanol/water (1:1),hexane, and ethyl acetate. The extracts were then used for qualitativeand quantitative  phytochemicalanalysis. The molecular study was performed with the DNA barcodingtechnique. The DNA was extracted from the root of the plant, and itspurity was examined by gel electrophoresis (1% w/v). The DNA was thenamplified using an Applied Biosystems 2720 thermal cycler for therbcL, matK, and ITS primers. The amplified primers were sequencedwith a 3130 Genetic Analyzer, and the generated sequences were searchedfor similarity in the GenBank Database using the nucleotide BLASTanalysis. The micro- and macroscopic studies revealed the morphologicaland organoleptic characters as well as the presence of medullary rays,fiber, cork, sclereids, parenchymal cells, and scalariform vessels.The physiochemical properties were found within the limit. The phytochemicalanalysis revealed the presence of terpenoids, flavonoids, saponins,and alkaloids. In addition, the alkaloidal content was high in theethanol extract (63.04 ± 3.08 mg At E/g), while the phenol contentwas high in the hexane extract (10.26667 ± 1.77 mg At E/g), andthe flavonoid content was high in the ethyl acetate extract (41.458± 1.33 mg At E/g). After the BLAST analysis from the GenBankdatabase, the rbcL, ITS, and matK primers showed a similarity percentageof 99.83, 99.84, and 100. The phylogenetic tree for the species closestto each primer was generated using the MEGA 6 software. The matK locihad the highest percentage similar to the rbcL and ITS loci, indicatingthat the matK loci can be used to identify the root of E. tithymaloides as a standalone. The results fromthis study can be used to establish a quality standard for E. tithymaloides that will ensure its quality andpurity.Adulteration and substitution of medicinal plants havebecome amatter of great concern in recent years.  This might rapidly increase the production ofmedicinal plant-based products such as teas, tinctures, ointments,and other forms of medicine, which will become more accessible toconsumers, leading to improved health outcomes for many individuals.This is important for a variety of reasons, such as being more cost-effectivethan other medicines, being organic and natural, and being readilyavailable in many parts of the world. In addition, these plants oftenhave fewer side effects than other treatments especially syntheticdrugs and heavy metal formulations.2The use of medicinal plantsfor treating various illnesses anddisorders has a long history, stretching back centuries. In recentyears, the use of medicinal plants in modern medicine has seen a rapidincrease due to their potential benefits in treating conditions likecancer, chronic pain, and other prevalent health problems.3 as well as in more recent sources like the British Pharmacopoeia.4 Authentication and identification of herbal ingredientsare necessary to address the problem of adulteration and substitution.With time, plants have become increasingly important to human medicaltreatments and advancements. The extinction of certain medicinal plantscan have a drastic effect on the continued growth of medical treatments,leading to a greater understanding of the importance of these plants.Without them, it is impossible to make progress in many areas of medicalresearch. The regulation of medicinal plants is a very important anddifficult task, which is why it is necessary to have stringent lawsto ensure safety and quality and to distinguish between the adulterationand substitution of medicinal plants.5 Despiteincreased regulations, it is hard to guarantee the safety and qualityof herbal products. This not only affects the people consuming theseplants but also adversely affects the environment as well. The majorpart of quality control (QC) is to check the product for any adulterationor contamination to determine its safety and efficacy. Generally,QC is done with analytical techniques, which involve the use of electrophoretic,6 chromatographic ,7 and hyphenated methods ,8 including specific and non-specific detectorsystems. Some other aspects that are included in QC are physical examination,microscopical examination, chemical examination, microbiological examination,stability testing, toxicological examination, and a combination ofthe aforementioned techniques.9 Despitethese advances, adulteration and substitutions persist in the currentperiod. These conventional pharmacognostic studies and techniques,even the use of modern hyphenated analytical systems, also failedto detect adulterants and substitutions.As the medicinal plant industry continues to expand with betterprofits, substitution and adulteration of herbal ingredients havebecome more and more of an issue. These practices are documented inancient medical texts, such as the Ebers Papyrus,11 There areseveral techniques available, including conventional sequencing, non-Sangersequencing, random amplifiable polymorphic DNA, DNA barcoding, amplifiablefragment length polymorphism, microsatellites, single nucleotide polymorphism,and others. The limitation with a few of these methods is that noideal marker exists for all the species, and sometimes, it may misleadin identifying the taxonomy but could be highly beneficial in populationgenetics.12 Because of its accuracy andreliability to identify variations, DNA barcoding has gained the mostpopular of all methods. Traditional methods of plant identification,such as morphological analysis, can be subjective and prone to error,particularly in cases where species are closely related or have similarphysical characteristics.14 On the other hand,DNA barcoding enables accurate identification of a plant species basedon its distinct genetic make-up. Since every plant has a differentDNA sequence, it includes using a short, standardized DNA sequenceas a “barcode” to identify a certain species of plant.The chloroplast gene rbcL, matK, and trnH-psbA are the most frequentlyused barcode region, but the ITS  markerhas been also used in recent studies.16 In a study,the ITS marker was used for DNA barcoding of Euphorbiaspecies, and the researchers found that this marker was able to effectivelydistinguish between Euphorbia subgenusand closely related species. It was reported that the ITS marker mightbe used to identify Euphorbia speciesand to verify the authenticity of the plant material used in conventionalmedicine and the pharmaceutical industry.17 Similarly, Akilabindu in 2019 used chloroplast rbcL and matK genefrom Flacourtia inermis Roxb for barcoding.It was reported that rbcL gene showed 100% similarity and matK geneshowed 99.20% similarity with Flacourtia jangomas.18 These studies strongly suggest thatmatK, ITS, and rbcL markers are intensively used in plant DNA barcoding.Despite being a promising technique, employing DNA barcoding for theauthentication and identification of medicinal plants comes with anumber of limitations such as limited reference libraries, inter-and intra-specific variation, and quality of plant material, cost,and complexity.19On the other hand,molecular biology has had a significant impacton plant science research, allowing us to uncover many previouslyunknown genetic and behavioral traits in plants. These genetic traitsare very helpful in identifying plants of particular species in casesof adulteration and substitution.Euphorbia tithymaloides, also knownas “devil’s-backbone” or “coast spurge,”is a medicinal plant that belongs to the family Euphorbiaceae. Itis widely distributed in Central and South America, including theAfrican and Caribbean. The plant is found in many other subtropicaland tropical areas as an invasive species. The plant is traditionallyused in folk medicine to treat various ailments, such as inflammatoryconditions, fever, and tumors. Some studies have also shown that extractsof E. tithymaloides have pharmacologicalactivities such as anti-inflammatory, anti-tumor, anti-diabetic, anti-cancer,anti-leishmanial, anti-malarial, anti-helminthic, anti-microbial,anti-oxidant, anti-ulcerogenic, and cytotoxicity.21 Extensive and elaborative research works have been carried out inthe aerial parts of the plant, while the roots are yet to be explored.Hence, this study aimed to carry out the pharmacognostical study of E. tithymaloides root by both conventional and molecularmethods.22.1E. tithymaloides were collected from Jamia Hamdard Herbal Garden, New Delhi, India.Department of Botany, Jamia Hamdard, New Delhi, India, helped to identifyand authenticate the plant. For future reference, a sample of theplant material was deposited in the herbarium under the voucher specimennumber BOT/DAC/2022/01. The roots were cleaned with water to removemud, broken into small pieces, and completely air-dried. The powderwas then pulverized into a coarse powder using an analytical millingmachine and stored in an airtight glass jar for use in the currentwork.In March 2022, root parts of 2.2E. tithymaloides wereexamined using visual perception. The color, odor, and taste of theroot were observed and recorded as organoleptic properties. The macroscopiccharacteristics of the root, such as shape, size, fracture, and othersurface features, were observed using the protocol mentioned in Indianpharmacopeia.22Thefresh roots of 2.3500 mgof a moderately fine (44/85) grounded root powder was immersed in10 mL of water (1:20) and left to stand overnight for 24 h. Subsequently,the contents were put into a Petri-plate, and a slide was preparedby placing the contents with a brush on a clean and dry slide. Thecontents were then examined using a Motic microscope moticam 3.0 MP,AE 2000, and images were taken.2.422 and WHO.23To assess the purity and quality of the crude drug, analytical standardsand physicochemical constants of the root were determined. The ashvalue , foamingindex, foreign matter, swelling index, moisture content, and extractivevalue  of root powderedwere determined using the standard protocol provided by the IndianPharmacopoeia2.5The extractswere prepared by using the Soxhlet apparatus by increasing the polarityof a solvent such as ethanol/water (1:1), ethyl acetate, hexane, andethanol. For each solvent, 2 h was given for extraction with optimaltemperature.2.62.6.124To investigate the existence of different secondarymetabolites in the crude extract, qualitative phytochemical assayswere carried out using established methods. Saponin test with frothtest; terpenoid/steroid test with Liebermann–Burchard reagent;flavonoid test with Shinoda test; Tannin test with ferrous(III) chloride;and alkaloid test with Dragendorff reagents.2.6.2The quantitative phytochemical tests were doneusing standard procedures to detect the number of secondary metabolitessuch as alkaloid, flavonoid, and phenolic compounds in the crude extract.2.6.2.125 Standard quercetin solutions of 20, 40, 60,80, and 100 g/mL were produced in 96% ethanol. The standard extractsolution (1 mg/mL) was prepared. 12 μL of 10% aluminium chloridesolution, 60 μL of extract solution, 180 μL of 96% ethanol,and 12 μL of 1 M sodium acetate were added to the mixture ina 96-well plate. 96 percent ethanol was used as a reagent blank. Afterbeing mixed, each substance was incubated for 40 min at room temperaturein a dimly lit area. The absorbance at 415 nm was measured with amicroplate reader. Total flavonoid content was calculated as quercetinequivalents per gram of plant extract.Total flavonoid content was determined using a slightly modifiedversion of the Sembiring et al. aluminium chloride colorimetric test.2.6.2.225 Ina flat-bottom microplate, a standard solution of extracts (1 mg/mL)was mixed with the Folin–Ciocalteu reagent (1:4) in a 40 and400 μL ratio and shaken for 2 min. After 5 min, sodium carbonatesolution (100 g/L) was added (125 μL) and shaken for 1 min ata medium speed. Absorbance was measured at 765 nm using a VersaMaxAbsorbance Microplate Reader after 3 h at room temperature. The absorbancewas corrected by subtracting the ethanol control reaction from thesample reaction. Gallic acid in dilutions of 10, 50, 100, 150, and200 μg/mL was used as calibration standards. The total phenoliccontent was expressed in milligrams of gallic acid equivalents (GAE)per gram of plant extract.The Folin–Ciocalteu method for a 96-well microplate wasoptimized based on Sembiring et al.2.6.2.326 Bromocresol green solution wasmade by dissolving 69.8 mg of BCG in 3 mL of 2 N NaOH and 5 mL ofdistilled water and then diluted to 1000 mL with distilled water.Phosphate buffer (pH 4.7) was prepared by adjusting the pH of 2 Msodium phosphate solution to 4.7 with 0.2 M citric acid. Atropinestandard solution was made by dissolving 1 mg of pure atropine in10 mL of distilled water. The extract was dissolved in 2 N HCl, filtered,and washed with CHCl3 (three times) and neutralized with0.1 N NaOH. 5 mL of BCG and 5 mL of phosphate buffer were added andcompletely extracted and diluted with chloroform.Accurately measured aliquots of atropine standard  were mixed with BCG and phosphate buffer, extractedwith chloroform, and diluted with chloroform. The complex absorbancein chloroform was measured at 470 nm using a UV-Spectrophotometer.The blank was prepared similarly but without Atropine.The total alkaloid content was determined using the method by Ajanalet al.2.72.7.1The NucleoSpin PlantII, Macherey-Nagel kit instructions were followed to extract DNA fromthe plant root sample (13817). The gel electrophoresis (1% w/v) methodwas used to ensure the purity of the extracted DNA and the same wasdocumented using the Bio-Rad.2.7.2Master Mix PhirePlant (Thermo Scientific) and Applied Biosystems 2720 thermal cyclerwas used for the amplification of the primers selected 1. PCR miExoSAP-IT PCR Product Cleanup Reagent (Thermo Fisher)was usedto clean the PCR product. Prior to sequencing, 2% agarose gel electrophoresiswas used to validate the PCR product’s purity. As a molecularstandard, a 100 bp DNA ladder  was employed. Usingthe BIO-RAD GelDoc-XR gel documentation system, gel images were captured.2.7.3The DNA sequencing wasperformed with the PCR products purified with the ExoSAP. ABI BigDyeTerminator v3.1 Cycle Sequencing reaction kit was used.2.7.4The 3130Genetic Analyzer Automated DNA Sequencing Machine were used to generateDNA sequences in.ab1 and FASTA formats, and Sequencing Analysis 5.1software was used to do further analysis. A contig of the truncatedsequence was created using forward and reverse strand sequences. Consequently,a single FASTA sequence was produced and further investigated. Usingthe nucleotide BLAST analysis tool, the sequencing similarity of thegenerated samples was examined with the sequences in the GenBank Database.The Clustal W alignment was utilized for multiple sequence alignmentand comparing different sequences, and the degree of similarity betweenthem was determined. For all three of the sample sequences, a phylogenetictree was constructed using the MEGA 6 software utilizing the closest-matchingsource sequence data from the database (NCBI GenBank nucleotide sequence).33.1The macroscopiccharacteristics of the roots as shown in 3.2E. tithymaloides (L.) Poit that are diagnostically important.3.3E. tithymaloides were determinedand are reported in Total ash, watercontent, alcohol soluble extractive, water-soluble extractive value,acid insoluble ash value, soluble ash, and moisture content of theroot of 3.4The extracts were studied further to discoverwhich phytochemical compounds were present. Flavonoid, alkaloid, terpenoids,steroid, tannin, and saponins are examples of common phytochemistrycomponents found in the root extract 4.3.53.5.1y = 0.043x + 0.122, R2 = 0.9947. Among the four crude extracts, ethyl acetate containedthe highest amount of total flavonoid content compounds followed byhexane, ethanol, and then hydro alcoholic   4. Among  0.9987) 5.3.5.3y = 0.0302x + 0.171 R2 = 0.996)  5. Of all= 0.996) 5.3.6Pedilanthus tithymaloides chloroplast rbcL gene (AB267959.1)showed a maximum similarity of 99.83 , the proportion of duplicate trees in which the linkedtaxa grouped together is displayed next to the branches.28 The tree is rendered to scale with branch lengthsin the same units as the evolutionary distances used to estimate thephylogenetic tree. The evolutionary distances, which are measuredin terms of the number of base substitutions per site, were calculatedusing the maximum composite likelihood technique.29 Six nucleotide sequences were subject to this investigation.Codon positions 1st + 2nd + 3rd + noncoding were included. For eachsequence pair, all uncertain positions were eliminated (pairwise deletionoption). The final dataset had 671 positions altogether. In MEGA X,evolutionary studies were carried out.30 The closest plant species was used to draw the phylogenetic treefor the matches in the BLAST data .The final dataset had 671 locations altogether. In MEGA X, evolutionarystudies were performed.30 From the BLASThits, the distance matrix showed the closest distance between thenearby species   8. The phtermined 9. The fi of 100% 10, follo plotted 11.3.6.1aDNA sequencingThe following sequences were generated for the sample(13817-R(rbcl)):>13817-R(rbCL-F) ATATTGGATCAAGCTGGTGTTAAGATTATAAATTGACTTATTATACTCCTGAATATGAAACCAAAGATACTGATATCTTGGCAGCATTCCGAGTAACTCCTCAACCTGGAGTTCCACCTGAGGAAGCAGGAGCTGCCGTAGCTGCTGAATCTTCTACTGGTACATGGACACTGTGTGGACCGATGGGCTTACCAGTCTTGATCGTTATAAAGGACGATGCTACCACATCGAGCCCGTTGCTGGAGAAGAAAATCAATATATTGCTTATGTAGCTTACCCCTTAGACCTTTTTGAAGAAGGTTCTGTTACTAACATGTTTACCTCCATTGTGGGTAATGTATTTGGGTTCAAAGCCCTACGCGCTCTACGTCTGGAGGATTTACGAATCCCTACTTCTTATACTAAAACTTTCCAAGGGCCACCTCATGGCATCCAAGTTGAGAGAGATAAATTGACAAATATGGTCGCCCTCTATTGGGTTGTACTATTAAACCAAAATTGGGGCTATCCGCTAGAATTACGGTAGAGCGGTTTATGAATGTCTTCGCGGTGGATTGAATTATTTCCAGA.>13817-R(rbCL-R) TAAGGCAACCCCAAAACAGAGACTAAAGCAAGTGTTGGATTCAAGGCTGGTGTTAAAGATTATAAATTGACTTATTATACTCCTGAATATGAAACCAAAGATACTGATATCTTGGCAGCATTCCGAGTAACTCCTCAACCTGGAGTTCCACCTGAGGAAGCAGGAGCTGCCGTAGCTGCTGAATCTTCTACTGGTACATGGACAACTGTGTGGACCGATGGGCTTACCAGTCTTGATCGTATAAAGGACGATGCTACCACATCGAGCCCGTTGCTGGAGAAGAAAATCAATATATTGCTTATGTAGCTTACCCCTTAGACCTTTTTGAAGAAGGTTCTGTTACTAACATGTTTACCTCCATTGTGGGTAATGTATTTGGGTTCAAAGCCCTACGCGCTCTACGTCTGGAGGATTTACGAATCCCTACTTCTTATACTAAAACTTTCCAAGGGCCACCTCATGGCATCCAAGTTGAGAGAGATAAATTGAACAAATATGGTCGCCCTCTATTGGGTTGTACTATTAAACCAAAATTGGGGCTATCCGCTAAGAATACGTAGAGCGTA.>13817-R(rbCL) (assembled contig) TAAGGCAACCCCAAAACAGAGACTAAAGCAAGTGTTGGATTCAAGGCTGGTGTTAAAGATTATAAATTGACTTATTATACTCCTGAATATGAAACCAAAGATACTGATATCTTGGCAGCATTCCGAGTAACTCCTCAACCTGGAGTTCCACCTGAGGAAGCAGGAGCTGCCGTAGCTGCTGAATCTTCTACTGGTACATGGACAACTGTGTGGACCGATGGGCTTACCAGTCTTGATCGTTATAAAGGACGATGCTACCACATCGAGCCCGTTGCTGGAGAAGAAAATCAATATATTGCTTATGTAGCTTACCCCTTAGACCTTTTTGAAGAAGGTTCTGTTACTAACATGTTTACCTCCATTGTGGGTAATGTATTTGGGTTCAAAGCCCTACGCGCTCTACGTCTGGAGGATTTACGAATCCCTACTTCTTATACTAAAACTTTCCAAGGGCCACCTCATGGCATCCAAGTTGAGAGAGATAAATTGAACAAATATGGTCGCCCTCTATTGGGTTGTACTATTAAACCAAAATTGGGGCTATCCGCTAAGAATTACGGTAGAGCGGTTTATGAATGTCTTCGCGGTGGATTGAATTATTTCCAGA.bBLAST analysisLibrary details: molecule type: DNA, query length: 607bases databasename: nr.cDistance matrixDescription: nucleotide collection (nt) *program:BLASTN 2.2.28+.3.6.2aDNA sequencing.The following sequences were generated for the sample(13817—ITS).>13817(ITS1) GGGCCGTAGCTACTGCAACGACCCGTGACATGTTCATAAACATGGGTGCCGGTGCGGGATTCGTCCGGCAACGGCATCCCACATGGGCCGGGCAGCGGGGACGCGGGTGGTGCACCCCGTGTTCTCCTTGTCCGGTTCCTTCTAACCAAACACCGACGCCAAACGCGTCAAGGAACTGCGAAAAAAAGGCAGCTTAGGCCCCGGAAACGGCGGTAACCAATGCTGTTTTGGAATAAAAACGACTCTCGGCAACGGATATCTCGGCTCTCGCATCGATGAAGAACGCAGCGAAATGCGATACTTGGTGTGAATTGCAGGATCCCGCGAACCATCGAGTCTTTGAACGCAAGTTGCGCCCGAAGCCTTTCGGCCGAGGGCACGCCTGCCCTGGGTGTCACTCAACTGTCGCCCCGACCCCCTCCTGAAAGGAGGGACGTGAGGGGCGGATGATGGCTTCCCGTGAGCTTTGCAGCCCGCGGTTGGCCCAAAATTCTGGTCCCTGGAACAGAATCCACGGCAATCGGTGGGTTGAAAGACCCTCGTAAAATGTCGTGGTTTCACGGAAGCCAGAGGCGGCAATTAGACCCCAGAGCGCATCCCAAAGCAGCGCTCGAACGGCGACCCCAGGTCAGGCGGGATTACCCGCTGAGTTTAAGCTTCGAGGGGGGGGGGGAGAGAAAAAA.>13817(ITS4) CTTTTTTTCGGCAGTTCCCGACGCCTTGGGTCGGGTTGGTAGAGACCGACAAGAGGAACACGGTGCACACCGGTCCCGCGCCCGCCCTTGGAGCCGTGCCGGACGATCCCGACCGGAAGCCAGTTTATGAAATGTCCCGGGTCGTTTGCGGGCGGGCCTGACAATGATCCTTTCGTAAGGGGGGGGCTGCGAAGGATCATGTCGAGGCCTGCCTAGCAAAACGACCCGTGAACATGTTCATAAACATGGCTGCCGGTGCGGGATTCGTCCGGCAACGGCATCCCCACATTGGCCGGGCAGCGGGGACGCGGGTGGTGCACCCCCGTGTTCTCCTTGTCCGGTTCCTTCTAACCCAAACACCGACGCCAAACGCGTCAAGGAACTGCGAAAAAAAGGCAGCTTAGGCCCCGGAAACGGCGGTAACCAATGCTGTTTTGGAATAAAAACGACTCTCGGCAACGGATATCTCGGCTCTCGCATGATGAAGAACGCAGCGAAATGCGATACTTGGTGTGAATTGCAGGATCCCGCGAACCATCGAGTCTTTGAACGCAAGTTGCGCCCGAAGCCTTTCGGCCGAGGGCACGCCTGCCTGGGTGTCACTCAACTGTCGCCCCGACCCCCTCCTGAAAGGAGGGACGTGAGGGGCGGATGATGGCTTCCCGTGAGCTTTGCAGCCCGCGGTTGGCCCAAAATTCTGGTCCCTGGAACAGAATCCACGGCAATCGGTGGTTGAAAGACCCTCGTAAAATGTCGTGGTTTCACGGAAGCCAGAGGCGGCAATTAGACCCCAGAGCGCATCCAAAGCAGCGCTCGAACGGCGACCCCAGTCAGGCGGATAGACCA.>13817-ITS(assembled contig) GGGTGCCGGTGCGGGATTCGTCCGGCAACGGCATCCCACATGGGCCGGGCAGCGGGGACGCGGGTGGTGCACCCCGTGTTCTCCTTGTCCGGTTCCTTCTAACCAAACACCGACGCCAAACGCGTCAAGGAACTGCGAAAAAAAGGCAGCTTAGGCCCCGGAAACGGCGGTAACCAATGCTGTTTTGGAATAAAAACGACTCTCGGCAACGGATATCTCGGCTCTCGCATCGATGAAGAACGCAGCGAAATGCGATACTTGGTGTGAATTGCAGGATCCCGCGAACCATCGAGTCTTTGAACGCAAGTTGCGCCCGAAGCCTTTCGGCCGAGGGCACGCCTGCCCTGGGTGTCACTCAACTGTCGCCCCGACCCCCTCCTGAAAGGAGGGACGTGAGGGGCGGATGATGGCTTCCCGTGCGCTTTGCAGCCCGCGGTTGGCCCAAAATTCTGGTCCCTGGAACAGAATCCACGGCAATCGGTGGTTGAAAGACCCTCGTAAAATGTCGTGGTTTCACGGAAGCCAGAGGCGGCAATTAGACCCCAGAGCGCATCCAAAGCAGCGCTCGAACGGCGACCCCAGGTCAGGCGGGATTACCCGCTGAGTTTAAGC.bBLAST analysis.Library details: molecule type: DNA, query length: 612bases databasename: nr.cDistance matrix.Description: nucleotide collection (nt) *program:BLASTN 2.2.28+.3.6.3aDNA sequencing.The following sequences were generated for the sample(13817M(matK)):>13817-M(MatK-F) ATAAGAGAAATTTCCGCATTTAATTATGTATCAGATGTATTAATACCTTATCCCATCCATCTTGAAAAATTGGTCCAAACCCTTCGTTTTTGGGTGACAGACCCTTCTTCTTTGCATTTTTTACGATTCTTTCTTCATCAGTATTGGAATTGGAACAGTCTTATTATTCCAAAGAAATCAATTTCGATTTTTCGAAAAAATAATCCACGATTTTTCTTGTTCATATATAATATTCATATATATCAATATGAATCCATCTTCTTTTTTCTTCGTAATCAGTCCTTTCATTTACGATCAACATTTTCTCGAGTCTTTCTTGAACGAATTTTTTTCTATGGAAAACTAGAACATTTTGCAGAAGTTTTTGCTAATGATTTTCAGACCATCCTAGGGTTGTTCAAGGAGCCTTTCATGCATTATGTTAGATATCAAGGAAAATCAATTCTGGCTTTAAAAGATAAGCCCTTTCTGATGAAAAAATGGAAATATTACCTTGTCAATTTATGTCAATGTCATTTTTATGTGTGGTTTCAACCAGAAAAGATCTATATCAATTCATTATCCAAAAATTCTCTCTATTTTGGGGGATATCTTTCAAGTGTACAAATCAATCCTTTGGTAGTACGGAGTCAAATGCTAGAAAATTCATATCTATAGCTAACGATGATACTATGAAGAAACTCGATACAATAGTTCCAATTACTCCTTTAATTAGATTATTGGCAAAATGCAATTTTGTAATGCAGTAGACATCCTATTAGTAAACCGATCCGGGCTCATTCATCCGATTCAGATATTATCGAACAATTTTGTGCGTATATGCAGAAATCTTTCTCATTATCTCGGGGGGGGGACTCACCAAAAA.>13817-M(MatK-R) CATCATCAAATATTTCCTTTTTAGAGGACAAATTTCCGCATTAATTATGTATCAGATGTACTAATACTATCCCATCCATCTTGAAAAATGGTCCAAACCCTTCGTTTTTGGGTGACAGACCATCTTCTTGCATTTTTTACGATTCTTCTTCATCAGTATTGGAATTGGAACAGTCTTATTATTCCAAAGAAATCAAATTTCGATTTTTCGAAAAAATAATCCACGATTTTTCTTGTTCATATATAATATTCATATATATCAATATGAATCCATCTTCTTTTTCTTCGTAATCAGTCCTTTCATTTACGATCAACATTTTCTCGAGTCTTTCTTGAACGAATTTTTTTCTATGGAAAACTAGAACATTTTGCAGAAGTTTTTGCTAATGATTTTCAGACCATCCTAGGGTTGTTCAAGGAGCCTTTCATGCATTATGTTAGATATCAAGGAAAATCAATTCTGGCTTTAAAAGATAAGCCCTTTCTGATGAAAAAATGGAAATATTACCTTGTCAATTTATGTCAATGTCATTTTTATGTGTGGTTTCAACCAGAAAAGATCTATATCAATTCATTATCCAAAAATTCTCTCTATTTTGGGGGATATCTTTCAAGTGTACAAATCAATCCTTTGGTAGTACGGAGTCAAATGCTAGAAAATTCATATCTAATAGCTAACGATAATACTATGAAGAAACTCGATACAATAGTTCCAATTACTCCTTTAATTAGATTATTGGCAAAAATGCAATTTTGTAATGCAGTAGGACATCCTATTAGTAAACCGATCCGGGCTCATTCATCCGATCAGATATATCGACAAATTTTGCCTATAATTCC.>13817-M(MatK)(assembled contig) TTATGTATCAGATGTATTAATACCTTATCCCATCCATCTTGAAAAATTGGTCCAAACCCTTCGTTTTTGGGTGACAGACCCTTCTTCTTTGCATTTTTTACGATTCTTTCTTCATCAGTATTGGAATTGGAACAGTCTTATTATTCCAAAGAAATCAATTTCGATTTTTCGAAAAAATAATCCACGATTTTTCTTGTTCATATATAATATTCATATATATCAATATGAATCCATCTTCTTTTTTCTTCGTAATCAGTCCTTTCATTTACGATCAACATTTTCTCGAGTCTTTCTTGAACGAATTTTTTTCTATGGAAAACTAGAACATTTTGCAGAAGTTTTTGCTAATGATTTTCAGACCATCCTAGGGTTGTTCAAGGAGCCTTTCATGCATTATGTTAGATATCAAGGAAAATCAATTCTGGCTTTAAAAGATAAGCCCTTTCTGATGAAAAAATGGAAATATTACCTTGTCAATTTATGTCAATGTCATTTTTATGTGTGGTTTCAACCAGAAAAGATCTATATCAATTCATTATCCAAAAATTCTCTCTATTTTGGGGGATATCTTTCAAGTGTACAAATCAATCCTTTGGTAGTACGGAGTCAAATGCTAGAAAATTCATATCTAATAGCTAACGATAATACTATGAAGAAACTCGATACAATAGTTCCAATTACTCCTTTAATTAGATTATTGGCAAAAATGCAATTTTGTAATGCAGTAGGACATCCTATTAGTAAACCGATCCGGGCTCATTCATCCGATTCAGATATTATCG.bBLAST analysis.cDistance matrix.Library details: molecule type: DNA, query length: 728bases databasename: nr description: nucleotide collection (nt) *program: BLASTN2.2.28+.4E. tithymaloides, was selected for the pharmacognosticstudy. Primarily, the macroscopic analysis of plants aids in identifyingauthentic materials. The E. tithymaloides root is macroscopically seen to be light brown on the outside andbuff color on the inside, with a fibrous texture and dusty and mildlybitter flavors. Similar to this, one of the essential factors in pharmacopeiais the study of powder microscopy. The powdered roots of E. tithymaloides revealed the presence of scalariformvessels with a pitted bordered wall, radially cut medullary rays,a group of fragmented oval-shaped parenchyma cells with a thin wallcontaining starch, a thick wall with an oval to rectangular-shapedcork, group of sclereids with polygonal wall, elongated, blunt end,and thin wall fibers.A pharmacognostic studyof a plant involves the scientific examinationof the plant’s physical and chemical characteristics, as wellas its traditional uses. The goal of such a study is to establisha set of quality standards for the plant material that can be usedto ensure its authenticity and purity. One such plant, E. tithymaloides. It was reported that the ethanol,ethyl acetate, and hexane extracts showed the presence of steroids,triterpenes, saponins, tannin, and coumarin. It was observed thatthe leaves lack alkaloids, which makes the root of E. tithymaloides more advantageous for therapeuticuse than the leaves.31Preliminary phytochemical investigationof medicinal plants isthe initial step in the process of identifying and characterizingthe phytoconstituents present in a plant. The root powder was subjectedto extraction with solvents such as ethanol, ethanol: water, hexane,and ethyl acetate. These extracts were then used for the physiochemicaland phytochemical analysis, which indicated the presence of alkaloids,tannins, flavonoids, steroids, terpenoids, and traces of saponins.A similar study was conducted by Matisui et al. in 2017, where thephytochemical analysis was performed with the leaves of E.tithymaloides was found to be 7.5%, indicating thetotal amount of inorganic matter present in the plant. The water-solubleash value of 2% revealed the presence of water-soluble compounds suchas inorganic compounds, acids, and sugars. The hexane, alcohol, andhydro-alcohol soluble extractive values show the presence of polar-solublesolvents such as tannins, flavonoids, and alkaloids. Similarly, thefoaming index and moisture content were both found to be <100 and3.33%, indicating that no foaming agents and less moisture were presentin the root samples. The presence of moisture in the plant materialcan have a significant impact on the quality and stability of thephytoconstituents present in the material. Moisture serves as an idealmedium for the growth of bacteria and fungi, which can lead to thedegradation of the plant material and the loss of its medicinal properties.33 Additionally, moisture can also cause the hydrolysis and oxidationof moisture-sensitive phytoconstituents, such as alkaloids, flavonoids,and terpenoids, which can result in a decrease in their concentrationand effectiveness. These findings agree with the phytochemical analysis,which shows the presence of polar-soluble solvents and traces of saponins.Extractivevalues are a measure of the amount of certain activeor inert ingredients present in a drug. They are used to determinethe purity and potency of a drug and can help detect if a drug isexhausted or adulterated. The United States Pharmacopeia (USP) andthe European Pharmacopeia (EP) provide guidelines for the acceptablerange of extractive values for various drugs. Drugs that fall outsideof these ranges may be considered exhausted or adulterated and shouldnot be used. The total ash content of the root of 34 This study examined the total amount of flavonoids,phenols, and alkaloids. The aluminum chloride colorimetric test wasused to evaluate the sample’s total flavonoid content. Theassay revealed that the ethyl acetate extract contains the highestamount of flavonoids, while the hydro-alcoholic extract showed thelowest amount. The ethyl acetate extract’s high concentrationof flavonoids is comparable to the research conducted by Chávezet al. in 2022. It was reported that, when compared to hexane, water,and dichloromethane, the ethyl acetate extract of E.tithymaloides leaves contained a high amount of flavonoids.35 This indicates that E. tithymaloides in general, as a whole plant, possesses a good amount of flavonoids.Likewise, using gallic acid as a reference, the Folin–Ciocalteumethod was used to quantify the total phenol concentration of theroot extracts. It was clear from the assay that the hexane extractshad a higher phenolic content than other extracts. The total alkaloidalcontent assay showed that the ethanol extract had a high amount ofalkaloids.The primary goal of quantitative chemical analysis is to estimatethe amounts of the plant’s major phytoconstituents classes.Flavonoids, phenols, and alkaloids are three important classes ofphytoconstituents that are commonly found in medicinal plants. Theyare known for their medicinal properties and have been used in traditionalmedicine for centuries. Their presence in medicinal plants is oftenused as a measure of the plant’s quality and potency.36 In this study, all three markers were used forthe identification of E. tithymaloides. The sequence match with rbcl loci showed a match of 98.64 to 99.83%with the top five hits from the BLAST analysis. Similarly, the sequencematch with the MatK loci was from 96.25 to 100%. These results arein contrast with the phylogenetic study conducted by El-Banhawy, 2020in the genus Euphorbia.37 It was reportedthat the rbcl was the least successful and matK genes were not significantlydifferent in identifying the Egyptian Euphorbia. However, the resultsof this study reveal a good identity score of 99.84 and 100 for Euphorbia plant grown in India. The difference inthe identification of the genus using the rbcl and matk loci couldbe varied due to the geographical location and environment of theplant growth. These genetic and geography-based changes and theiridentification by rbcl and ITS were expressed by Shawkat and Ahmed,2022 in a comparative study. It was reported that the rbcl and ITS2were able to provide a good resolution among the Euphorbiatirucalli, Euphorbia hirta, and Euphorbia peplus but showedonly a minor change in the evolution taxa and phylogenetic tree.39 These data are comparable to the results observedin this study as the phylogenetic tree and the distance matrix showsa minor change and were able to identify the closest species witha good matching percentage. The ITS is considered to be one of themost informative markers for species identification in plants, asit has a high degree of variation among different plant species. TheITS region is located between the 18S and 28S rRNA coding regionsand is transcribed as part of the rRNA precursor molecule. The ITSregion is highly variable, containing both coding and non-coding regions,which makes it a valuable marker for species identification. Additionally,the ITS region is present in multiple copies in the genome, whichincreases the chances of detecting variations among different species.38 Likewise, the ITS primers ITS1 and ITS4 in thisstudy revealed a good match from 95.11 to 99.84% from the BLAST analysis.The ITS loci results are comparable with the study experimented onby Kim et al., in 2020 where DNA barcoding was performed for KoreanEuphorbiaceae. It was reported that among the rbcl, ITS, and MatKloci, the ITS was the most beneficial and can be used to identifyother Korean Euphorbiaceae plants using ITS as a single barcode.40 Likewise, the results from this study show thatall the three loci were able to efficiently identify the E. tithymaloides. In addition, the 100% true matchwith the MatK loci shows that it can be used as a standalone to identify E. tithymaloides from the subspecies and other Euphorbiaceaeplants as well.The nuclear ribosomal DNA ITS regions ITS1 and ITS2and the chloroplastgenes matK, rbcL, and trnH-psbA are the markers in plant barcodingthat are most often investigated. These markers are highly informativefor identifying and differentiating plant species. Recently, the useof DNA barcoding based on the markers rbcl, ITS, and MatK has gainedsignificant traction in the field of plant species authentication.E. tithymaloides is the high demand for the plantand its medicinal properties. This has led to the collection of theplant from wild populations, which can lead to over-harvesting anddepletion of wild populations. Another reason is that E. tithymaloides is often confused with closely relatedspecies, and this can lead to misidentification and substitution.For example, E. tithymaloides is oftenconfused with Euphorbia lathyris, whichis not used for medicinal purposes and is considered toxic. Adulterationand substitution can have serious consequences for both the consumersand the environment. Consumers may be unknowingly consuming harmfulsubstances or receiving ineffective treatment, while wild populationsof medicinal plants may become endangered due to over-harvesting.To address these issues, it is important to establish and use DNAbarcoding techniques for the identification and authentication ofmedicinal plants, including E. tithymaloides, to ensure that the plant material being sold is authentic and unadulterated.Additionally, conservation measures should be implemented to protectwild populations of medicinal plants from over-harvesting, and regulationsshould be put in place to control the trade of medicinal plants.One of the main reasons for the adulterationand substitution of 5E. tithymaloides has been carriedout in this study and reported for the first time. The results fromthe micro-and macroscopic studies, phytochemical analysis, and DNAbarcoding had shown significant results in identifying the E. tithymaloides. It is important to keep in mindthat DNA barcoding is one tool among many that can be used to identifyand authenticate medicinal plants and it should be used in combinationwith other methods, such as morphological and chemical analysis, toensure accurate identification. The results of this pharmacognosticalstudy can be used to establish a set of quality standards for the E. tithymaloides, which can be used to ensure itsauthenticity and purity. This is important for ensuring the safetyand efficacy of traditional medicine and medicinal products made from E. tithymaloides.The conventional and molecularpharmacognostic study on the rootof


The current study aims to investigate the regulatory impact of leptin or melatonin on bone metabolism as well as the underlying mechanism in conjunction with Sema4D .Rats were used to create the osteoporosis model utilizing the OVX (OVariectomize) technique. Rat tibial specimens from each side were collected for three-dimensional reconstruction and Micro-CT scanning examination. The Hematoxylin-osinstaining (HE) staining technique was used to determine the pathological condition of bone tissues. The ELISA (Enzyme-Linked Immunosorbent Assay) assay was used to measure the amount of estradiol present in the serum. In the current study, there were six groups: control, OVX, OVX + NL (no load group), OVX + Sema4D, OVX + Sema4D + leptin, and OVX + Sema4D + MT (melatonin). Rats were given injections of the Sema4D or leptin overexpressing vectors via the tail vein in accordance with the aforementioned classification. By using a high-resolution micro-CT technology, 3D bone structure was discovered. The activity of tartrate-resistant acid phosphatase-5b (TRAP-5b) and bone-derived alkaline phosphatase (BALP) in serum was assessed using an ELISA. The number of osteoclasts in the metaphysis of the upper tibia was determined using TRAP (tartrate-resistant acid phosphatase) staining. Immunohistochemistry was used to find leptin and bone morphogenetic protein-2 (BMP-2) expressions in bone tissue.The BV/TV (Bone volume/Tissue volume), Tb.N (Trabecular number), BMD , and BMC  levels were significantly higher in the OVX + Sema4D + leptin and OVX + Sema4D + MT groups compared to OVX + NL, while Tb.Sp (Trabecular separation) levels were significantly lower. In contrast to the OVX group, the bone trabeculae in the OVX + Sema4D + leptin and OVX + Sema4D + MT groups had a relatively complete structure and tended to be organized closely. The amount of bone trabeculae grew drastically, whereas the proportion of TRAP-positive osteoclasts declined dramatically. BMP-2 and leptin were also elevated, while BALP and TRAP-5b activity was reduced.Leptin or melatonin improved Sema4d's role in trabecular bone microstructure, bone production, and repairment of trabecular bone loss in osteoporosis rats. A prevalent age-related illness is osteoporosis. Osteoporosis has recently emerged as a major global public health concern due to the sharp rise in patient numbers . StatistSema4D, a protein involved in brain signaling, belongs to the semaphorin family. It has been established that semaphorins, also known as immunological semaphorins, and immune control have a close association. According to reports, Sema4D is involved in a wide range of signal transduction pathways and performs a wide range of biological functions, including the control of B cells, T cells, and other immune cells as well as the development of the nervous system, platelet function, epithelial cell function, and endothelial cell function . Sema4D Using an OVX rat model, the current work intends to investigate the effects and processes of Sema4D, Sema4D paired with leptin, and Sema4D combined with melatonin on bone metabolism in an effort to identify possible molecular targets for osteoporosis therapeutic treatment.SD (Sprague Dawley) rats, 32 females, 200–220 g Zhejiang Weilihua Experimental Animal Technology Co., Ltd., License No. SCXK (Zhejiang) 2019-0001), Rearing Environment: Temperature 20–26 °C, Humidity 40%- 70%. All research was done in compliance with international standards for the treatment and use of laboratory animals, and needless harm to the animals was avoided at all costs .Medicine refrigerator , refrigerated freezer , slicer , electric blast drying oven , thermostatic incubator , electric thermostatic incubator ; Pressure cooker , induction cooker , fume hood; Microscope , microtome , multifunctional enzyme marker ; First antibody: BMP-2 ; leptin; Sema4d ; Second antibody: horseradish enzyme-labeled goat anti-rabbit IgG (H + L) ; Tartrate-resistant acid phosphatase staining solution ; Rat tartrate-resistant acid phosphatase 5b (TRACP-5b) ELISA reagent ; Rat bone alkaline phosphatase (BALP) kit ; Melatonin (S20287) was purchased from Shanghai Yuanye Biotechnology Co., Ltd., with a relative molecular weight of 232.28 and a purity of more than 99%.Construct the PCR primer for the target fragment based on the sequence of the target gene Sema4D  and Leptin ; As the carrier for digestion, the suitable restriction enzyme was chosen, and the pure linear carrier was extracted from the agarose gel; Then, perform PCR on the target fragment and recover the agarose gel to acquire the right fragment size; Link the linearization vector to the target fragment utilizing homologous recombination or the T4 technique; Transformed responsive DH5a or stbl3, covered with bacterial fluid and cultured for 12 to 16 h; Choose the moving monoclonal colony for validation; Choose the most appropriate positive clone for colony verification prior to sequencing; The extraction of plasmids was carried out on cloned samples with the right sequence. Anhui General Biology Co., Ltd. is responsible for the adenovirus packaging of the vector after the sequencing verification. The specific information on the viral vector is as follows :ATGAAGATGTGTGCCCCCGTCAGGGGGCTGTTCTTGGCCCTGGTGGCTGTGTGGAGGACCGCGGTGGCATTCGCCCCTGTGCCTCGGATCACCTGGGAGCACGGAGAGGTAGGTCTGGTGAAGTTTCACGAGCCAGGCATCTTTAACTACTCTTCCTTGCTGATGAGTGAAGACAAAGACACTCTGTATGTGGGTGCCCGGGAAGCTGTCTTTGCAGTGAATGCGCTGGACATCTCTGAGAAGCAACATGAGGTATACTGGAAGGTCTCTGAAGACAAAAAATCCAAGTGCGCAGAGAAGGGGAAATCAAAGCAGACGGAGTGCCTTAACTACATCCGAGTGCTGCAACCGCTTAGCAGCACTTCCCTCTACGTGTGTGGGACCAATGCGTTCCAGCCCACCTGTGACCACCTGAACTTGACCTCTTTCAAGTTTCTGGGGAAAAGCGAAGATGGCAAAGGAAGATGCCCCTTCGACCCCGCCCATAGCTACACATCCGTCATGGTCGGGGGAGAGCTCTACTCTGGGACTTCATATAATTTCTTGGGCAGCGAACCCATCATCTCTCGAAACTCTTCCCACAGTCCCCTGAGGACAGAGTACGCCATCCCTTGGCTAAACGAGCCTAGCTTCGTCTTTGCTGACGTGATCCACAAGAGCCCAGATGGTACAGAGGCTGAGGATGACAAGGTCTACTTCTTCTTTACGGAGGTGTCCGTGGAGTACGAGTTCGTCTTCAAGTTGATGATCCCGCGAGTTGCCAGGGTGTGCAAGGGCGACCAGGGCGGCCTGCGGACTTTGCAAAAAAAGTGGACCTCCTTCCTAAAGGCCAGACTGATCTGCTCCAGGCCAGACAGTGGCCTGGTCTTCAACATTCTTCAAGATGTGTTTGTGCTGAGGGCCCCGGGCCTCAAGGAACCTGTGTTCTATGCGGTCTTCACCCCACAGCTGAACAACGTGGGTCTGTCAGCGGTCTGTGCCTACACGCTGTCCACGGTGGAGGCCGTCTTCTCCCGAGGAAAGTACATGCAGAGTGCCACAGTGGAGCAGTCTCACACCAAGTGGGTACGCTACAATGGCCCAGTGCCCACTCCCCGGCCTGGAGCGTGTATCGACAGTGAGGCCCGGGCAGCCAACTACACCAGCTCCTTGAATCTCCCAGACAAAACGCTGCAGTTTGTCAAAGACCACCCTTTGATGGACGACTCGGTGACGCCAATAGACAACAGGCCGAAACTGATCAAAAAAGATGTCAACTACACCCAGATAGTGGTAGACAGGACCCAGGCCCTGGATGGGACCTTCTACGACGTCATGTTCCTCAGCACAGACCGGGGCGCTCTGCATAAAGCTGTCATCCTTGCAAAAGAGGTACACGTGGTTGAGGAGACCCAACTCTTCCAGGACTTCGAACCGGTCCTGTCTCTGCTGCTATCATCAAAGAAGGGGAGGAAGTTTGTCTATGCTGGCTCCAACTCAGGAGTGGTCCAAGCTCCCCTGGCCTTCTGCGGAAAGCACAGTAGCTGTGAAGACTGTGTGCTAGCACGGGACCCCTACTGCGCCTGGAGCCCAGCCATCAAGGCCTGTGTTACCTTGCACCAGGCAGAGGGCTCTAGCAGGGGCTGGATTCAGGACATGAGTGGCGACACGTCCTCGTGCCTGGATAAGAGTAAAGAAAGTTTCCATCAGCATTTTTTCAAGCACGGCGGCACAGCAGAACTCAAGTGTTTCCAAAAGTCCAACCTGGCCCGGGTGGTGTGGAAGTTCCAGAACGGCGAGTTGAAGGCTGTGAGTCCCAAGTATGGCTTTGTGGGCAGGAAGCACCTGCTCATCTTTAACCTGTCAGACGGAGACAGCGGTGTGTACCAGTGCCTGTCAGAGGAAAGGGTCAGGAATAAAACGGTCTCCCAGCTGCTCGCCAAGCACATCCTGGAAGTGAAAATGGTAGCTCGGATCCCCCCATCACCTACCTCACAGACTGCTCAGACAGAAGGTAGTAGGATCACATCCAAAATGCCTGTGGCGTCTACCCAGGGGTCCTCTCCCCCTACCCCGGCTCTGTGGGCAACCTCCCCCAGGGCTGCCACCCTACCTCCCAAGTCCTCCTCCACCGGCACGTCCTGTGAACCAAAAATGGTCATCAACACGGTCCCACAGCTCCACTCGGAGAAGACAGTGTATCTCAAGTCCAGTGACAACCGCCTGCTCATGTCTCTCCTCCTCTTCCTCTTTGTCCTCTTCCTCTGCCTCTTTTCCTACAACTGCTACAAGGGCTACCTGCCCGGACAGTGCTTAAAGTTCCGCTCAGCCCTGCTGCTCGCAAAGAAAAAACCCAAGTCAGAGTTCTCTGACCTGGAGCAGAGTGTGAAGGAGACGCTGGTAGAACCTGGGAGCTTCTCGCAGCAGAACGGCGACCAGCCCAAGCCAGCCTTGGATACCGGCTATGAAACCGAGCAGGACACTATCACCAGCAAGGTCCCCACCGATCGAGAGGACTCGCAACGTATCGACGAGCTCTCCGCCAGGGACAAACCGTTTGATGTCAAGTGTGAACTCAAGTTTGCAGACTCGGATGCCGACGGGGACTGA.ATGTGCTGGAGACCCCTGTGCCGGTTCCTGTGGCTTTGGTCCTATCTGTCCTATGTTCAAGCTGTGCCTATCCACAAAGTCCAGGATGACACCAAAACCCTCATCAAGACCATTGTCACCAGGATCAATGACATTTCACACACGCAGTCGGTATCCGCCAGGCAGAGGGTCACCGGTTTGGACTTCATTCCCGGGCTTCACCCCATTCTGAGTTTGTCCAAGATGGACCAGACCCTGGCAGTCTATCAACAGATCCTCACCAGCTTGCCTTCCCAAAACGTGCTGCAGATAGCTCATGACCTGGAGAACCTGCGAGACCTCCTCCATCTGCTGGCCTTCTCCAAGAGCTGCTCCCTGCCGCAGACCCGTGGCCTGCAGAAGCCAGAGAGCCTGGATGGCGTCCTGGAAGCCTCGCTCTACTCCACAGAGGTGGTGGCTCTGAGCAGGCTGCAGGGCTCTCTGCAGGACATTCTTCAACAGTTGGACCTTAGCCCTGAATGCTGA.The following is the map of the Semad4 adenovirus overexpression vector:The vector map of Leptin adenovirus overexpression is as follows:The occurrence and progression of osteoporosis are significantly influenced by the estrogen released by the ovaries. Rats' decreased estrogen secretion following ovariectomy results in a weaker suppression of osteoclasts. Bone production is outpaced by bone absorption. Because the bone is in a high stage of transition, the mass of the bone cannot be balanced, which causes more bone to be lost. The best model to research postmenopausal osteoporosis is the ovariectomized osteoporosis model , 20; AccBai et al.  describeObtain serum samples and separate the supernatant for testing using a centrifuge. After treatment, BALP, TRAP-5b, and estradiol were identified according to the processes outlined in the instructions for the respective ELISA kit –27. Use Following the collection of tibia tissues from each animal, the tissues were washed in water for two hours. After dehydration with various concentrations of ethanol solution, tissues were dehydrated with xylene until translucent, followed by embedding for one hour and slicing. The slides were then roasted, dewaxed, hydrated, bathed in distilled water, colored in a hematoxylin aqueous solution for three minutes, and then differentiated using a hydrochloric acid ethanol differentiation solution for fifteen seconds. After being cleaned briefly with water and blue-returning solution for 15 s, slides were rinsed with water and stained with eosin  for three minutes. The inverted microscope was then utilized to capture images .Sections of paraffin were deparaffinized for 5 min, then progressively incubated in 100% ethanol for 5 min, 90% ethanol for 2 min, and 70% ethanol for 2 min. The TRAP staining solution was then added for fixation at 4 °C for 60 s. After being washed with water, slices were immersed in TRAP incubation solution at 37 °C for 45–60 min, then stained with methyl green for 2 to 3 min. Pictures were captured using a microscope .The slides were washed in PBS for one hour before being incubated overnight with 10% goat serum. The slides were then incubated with primary antibodies against BMP-2  or leptin  at 4 °C for 24 h, after which they were washed with PBS. The secondary antibody  was then added, followed by a 24-h incubation at 4 °C, rinsing, and DAB staining. Finally, photos were captured with an inverted microscope .For statistical analysis, SPSS 20.0 software was used. Each experiment was conducted three times. The mean standard deviation was used to express the quantitative outcomes (X As shown in Fig. As demonstrated in Fig. As shown in Fig. TRAP staining was used to evaluate the number of osteoclasts in metaphysis at the upper end of the tibia. Compared to control, the number of TRAP positive osteoclasts was dramatically increased in the OVX, OVX + NL, and OVX + Sema4D groups, among which the highest number of osteoclasts was observed in the OVX + Sema4D group. Furthermore, compared to the OVX group, dramatically declined TRAP positive osteoclasts were observed in the OVX + Sema4D + leptin and OVX + Sema4D + MT groups (Fig. As shown in Fig. As shown in Fig. Sema4D plays a crucial function in the regulation of osteoclast formation. Sema4D expression is increased during osteoclast proliferation ; NegishiIn the present study, OVX was used to construct an osteoporosis rat model. Pathological results showed that the number of tibial trabeculae in OVX rats was significantly reduced, and the bone trabeculae were thin and fractured with low density, accompanied by an increased serum E2 level. These results were consistent with the research reports of Zhao  and WangLeptin is reported to promote the proliferation of osteoblasts and the transformation of bone marrow stromal cells into osteoblasts. In previous experiments, an extremely repressed long-bone growth, reduced bone surface area, and decreased mineral and bone density level were observed in leptin deficient mice which were dramatically rescued by the injection of leptin, indicating that leptin played an important role in the differentiation and maturation of osteoblasts . It is rThe effect of melatonin on primary osteoporosis has been widely reported. St Hilaire et found that melatonin levels were lower in premenopausal women and were accompanied by increased bone resorption . Maria rIn conclusion, leptin and melatonin reversed the activation of osteoclasts caused by overexpression of Sema4D, which intensified the pathological process of osteoporosis. The target molecules for the clinical therapeutic development of osteoporosis were offered by the current study as potential and trustworthy candidates.


Pythium flevoense was diagnosed as the cause of dermatitis in a young adult female harbour porpoise (Phocoena phocoena) that had been trapped in a pound net in a temperate saltwater environment. Disease from Pythium sp. infection—pythiosis—is infrequently diagnosed in humans, horses, dogs, cattle, and few other mammalian species. Pythiosis is typically associated with exposure to tropical or subtropical freshwater conditions, and typically caused by Pythium insidiosum. However, until now, pythiosis has been reported in neither marine mammals nor temperate saltwater conditions, and P. flevoense is not known as a cause of pythiosis in mammals. This porpoise developed generalised dermatitis despite treatment and euthanasia was necessary. Histopathological evaluation revealed a chronic active erosive dermatitis, with intralesional hyphae morphologically consistent with a Pythium sp. PCR analysis and sequencing of affected skin matched Pythium flevoense with a 100% similarity to the reference strain. Additional diagnostics excluded other pathogens. Based on this case report, P. flevoense needs to be considered as a mammalian pathogen. Furthermore, harbour porpoises and possibly other marine mammals may be at risk of infection with P. flevoense, and pythiosis should be included in the differential diagnosis of dermatitis in marine mammals.The oomycete The online version contains supplementary material available at 10.1186/s13567-023-01226-1. Pythium species are oomycetes, fungus-like filamentous eukaryotic organisms belonging to the kingdom Stramenopila, phylum Oomycota, class Oomycetes, order Pythiales, family Pythiaceae, which may reproduce both sexually and asexually [Pythium insidiosum in humans and other animals may cause disease, called pythiosis (insidiosi), typically characterized by necrotising and granulomatous inflammation [sexually –3. Most sexually . Howeverammation , 3. The ammation , 3, suppammation .P. insidiosum occurs typically in (sub)tropical stagnant freshwater containing motile zoospores that enter skin lesions and invade tissues. Zoospores show tropism for skin and hair [Infection with and hair . Pythiosand hair –8. Pythiand hair , 3.Depending on the site of infection and involved species, cutaneous, lymphonodular, osseous, vascular, ocular, visceral, and disseminated forms of pythiosis may occur . VasculaP. insidiosum is morphologically discernible as hyaline, pauci-septate, nonparallel thin-walled (5–10 μm) hyphae  with infrequent and irregular branching. As routine H&E stains may fail to stain these hyphae, enhancement of discernibility requires silver stains like Grocott silver stain [Histologically, er stain , 15.Pythium flevoense was isolated and identified for the first time in soil samples from the province Flevoland, The Netherlands in 1968 [Parabroteas sarci) in Argentina [Plecoglossus altivelis) in Japan [P. flevoense, and simultaneously the first case of pythiosis in a marine mammal, a harbour porpoise (Phocoena phocoena). in 1968 . Sporadirgentina  and ayu in Japan . Here wend of May 2018, trapped in a pound net in the coastal seawaters near the town Korsør in Denmark. It was freed and brought to the Marine Biological Research Center, University of Southern Denmark, and intended to be part of the porpoise collection at the neighbouring sea aquarium Fjord & Bælt at Kerteminde Bay which is permitted by Danish law to house bycaught harbour porpoises for research and conservation purposes. It was named Idun and kept alone in a quarantined saltwater basin. A separated basin housed another normal healthy harbour porpoise. Both basins were circulated continuously with the same fresh seawater from the bay. Despite treatments for a progressive generalised erosive dermatitis, the animal did not recover and was euthanised after 3½ months . The carcass was stored on wet ice directly after euthanasia until autopsy was performed, 24 h after death.A female harbour porpoise, estimated to be 14 months of age, was found alive on the 22During autopsy the porpoise’s external and internal organs were examined. The skin lesions were photographed and sampled for histology, bacteriology and molecular analysis. Internal organs and tissues were similarly sampled. Samples for histology of skin, lung, trachea, heart, kidney, spleen, lymph nodes, urinary bladder, liver, pancreas, stomach, small and large intestines were fixed by immersion in 10% neutral-buffered formalin. These fixed samples were routinely processed and embedded in paraffin. Tissue sections of 4 μm thick were mounted on glass slides, deparaffinised with xylene, rehydrated using graded alcohols, and stained according to routine histochemical protocols  with haeSamples from affected skin were biopsied from the live animal on day 34 after rescue and taken during autopsy on day 105 after rescue. Samples for molecular analyses were stored in lysis buffer until processing. Tissue samples for bacteriology including affected skin were taken during autopsy on day 105, stored and frozen at −20 °C and subsequently thawed a week later for further analyses. Fluorescent microscopy using Blancophor was performed on the skin samples (taken on day 34), to visualize fungal(-like) organisms. Fungal cultures from the same samples were performed using SGA agar  and YGC agar  with incubation at 35–37 °C for 2 weeks. Species identification included classical techniques including macro- and micromorphology and thermotolerance testing. Molecular analysis included internal transcribed spacer (ITS)-PCR and sequencing of which the results were compared with online sequence databases in July 2018, NCBI (BLAST) and CBS* database. . The sequence analysis was repeated in August 2021 specifically for this study.For bacteriological examination, samples of skin, lung, kidney, liver, spleen, uterus, tracheobronchial lymph node and adrenal gland were frozen at −20 °C and after thawing, cultured according to an in-house established standard protocol, including classical methods and commercial API identification kits , for bacterial species from marine mammals, as described previously . For virnd of May 2018 into the Marine centre (day 0), blood samples were collected. Results from standard haematological and clinical biochemical analyses on these samples were all within normal ranges, without any indication of immunosuppression, anaemia, or inflammation  , days 3 to 29: enrofloxacin , days 20 to 104: tramadol , days 25 to 31: itraconazole , days 27 to 37: miconazol , days 29 to 49: terbinafine , days 31 to 45: posaconazole , days 45 to 101: voriconazole , days 45 to 104: amoxycillin with clavulanic acid , days 100 to 102: posaconazole , days 100 to 103: terbinafine , days 101 to 103: enilconazole (14.4% w/w baths), days 103 to 104: enrofloxacin .Macroscopic examination showed a female non-pregnant harbour porpoise in moderate physical condition , with a generalised dermatitis. The dermatitis was characterised by multifocal to coalescing lesions with an irregular rough surface , threadlike structures were discernible. These structures did not resemble true hyphae. They lacked the usual characteristics of fungal hyphae since they were of significantly variable wideness, had no distinct regular septa, and the tips were cut rather than rounded hyphal ends and fluorescence was not distinct as is usual for fungal hyphae.Aspergillus niger on YGC agar only. As A. niger is found ubiquitously as cause of black mold on certain fruits and vegetables, this single culture growth is considered more likely as a contaminant rather than a primary pathogen.After 2 weeks, fungal culture of one of four skin samples (taken on day 34) grew Mucorales sp. (zygomycetes) and for Aspergillus sp. were all negative (likely confirming contaminated growth of Aspergillus niger in culture), but all four samples were positive for Pythium sp. The results of ITS sequencing in 2018 matched Pythium pectinolyticum with a similarity of > 99.5% compared with the reference strain CBS* 122643 . Sequence analysis was rerun in August 2021 for the current study, and this time showed a 100% sequence similarity to CBS strain 234.72 of Pythium flevoense, which had been submitted to the database after 2018, and thus the aetiological species was reclassified as P. flevoense in this study. The fasta of the sequenced porpoise skin samples were:Specific fungal PCRs on four skin samples (taken on day 34) for >ITS1&2GCCCGTCGCACCTACCGATTGAATGACTCGGTGAGAAATCGGGACCGTGAATCCGTTTGCTTCATTGCGAGTGGACTTATGGGAACTTTTTCTAACCTCGCCATTTAGAGGAAGGTGAAGTCGTAACAAGGTTTCCGTAGGTGAACCTGCGGAAGGATCATTACCACACCCTAAAACTTTCCACGTGAACCGTTGTAAATATGTTCTGTGCTCTCTCTCGGGAGAGCTGAACGAAGGTGGCCTGCTTAATTGTAGACTGCCGATGTACTTTTAAACCCATTAAACTAATACTGAACTATACTCCGAAAACGAAAGTCTTTGGTTTTAATCAATAACAACTTTCAGCAGTGGATGTCTAGGCTCGCACATCGATGAAGAACGCTGCGAACTGCGATACGTAATGCGAATTGCAGAATTCAGTGAGTCATCGAAATTTTGAACGCACATTGCACTTTCGGGTTATGCCTGGAAGTATGCCTGTATCAGTGTCCGTACATCAAACTTGCCTTTCTTTTTTTGTGTAGTCAAGATTAGAAACGGCAGACTGTGAGGTGTCTCGCTGACTCCCTCTTCGGAGGAGAAGACGCGAGTCCCTTTAAATGTACGTTCGCTCTTTCTTGTGTTTAAGTAGAAGTGTGACTTTCGAACGCAGTGATCTGTTTGGATCGCTTTGCTCGAGTAGGCGACTTCGGTTAGGACATTAAAGGAAGCAACCCTATTGGCGGTATGTTAGGCTTCGGCCCGACTTTGCAGCTGACGGTGTGTTGTTTTCTGTTCTTTCCTTGAGGTGTACCTGTCTTGTGTGAGGCAATGGTCTaGGCAAATGGTTATTGTGTAGTAGGAAGTTGCTGCTCTTGAACGCCCTGTttTCGGATAGGGTAAAGGAGGCAACACCAATTTGGGATAGTCTTTGATTTATCATTGGCGCTCTTTCTAATTGGACCTGATATCAGGCAAGACTACCCGCTGAACTTAAGCATATTAATAAGCGGAGGAAAAGAAACTAACAAGGATTCCCCTAGTAACGGCGAGTGAAGCGGGATGAGCTCAAGCTTAAAATCTCTGTGCCAGTTTGGCATGGCGAATTGTAGTCTATGGAGGCGCTATCAGTGCGATTGTTCGGGG.Vibrio parahaemolyticus, considered a bacterial commensal of harbour porpoise’s skin flora. Bacteriological cultures (of samples taken during autopsy on day 105) were unremarkable, with no growth obtained after 14 days of culture in all sampled sites, except for a few Pseudomonas sp. from the tracheobronchial lymph nodes and also from skin, along with a few Acinetobacter sp.Bacteriological PCR analysis of the affected skin samples (taken on day 34) were positive for Virological PCR analysis on samples from brain, lung, kidney and urinary bladder collected during autopsy on day 105 tested negative for the presence of morbilliviral RNA.Pythium insidiosum as the causative pathogen [P. flevoense infection as the cause of dermatitis in a harbour porpoise. To our knowledge, it is the first time both for the diagnosis of pythiosis in a marine mammal and for P. flevoense as a cause of disease in a mammalian species. P. flevoense infections were reported previously in non-mammalian species only; ayu fish larvae [Pythium species as the causative pathogen was indicated by histopathological evaluation and confirmed by DNA sequencing that identified this particular, pathogenic species. Histological examination, especially in silver stains, revealed intralesional presence of fungus-like hyphae of approximately 7–10 μm diameter with rare septations and irregular branching  were detected increasingly at warmer freshwater temperatures >20 °C in Argentina [Pythium species have been isolated from seaweeds and algae in temperate coastal saltwater environments [P. flevoense infection was reported in ayu fish larvae reared in artificial saltwater conditions at 12–15 °C in a fishery in Japan [Another unusual aspect of this case was the temperate saltwater environment, which is in contrast with the subtropical stagnant freshwater conditions, such as swamps and rice fields, where infections with ntil now , 29, 30.rgentina . Nonetheronments , 32. Morin Japan . Even soP. flevoense in the water. Skin wounds in animals and humans are reported to serve as potential port d’entrée for the infectious motile zoospores [P. flevoense was isolated from soil samples previously, another possible explanation is that the seawater in the bay and basin may have been contaminated via freshwater runoff from land. However, another harbour porpoise that was kept in a separated adjacent basin circulated with the same seawater remained healthy without any signs of dermatitis or skin lesions.As pythiosis is not known to be contagious between mammals, infection in our case most likely occurred at sea or in the marine centre, presumably from infectious zoospores of oospores . In our oospores , 34. Furoospores , 32. TheImmunosuppression and hemopathies are risk factors for contracting pythiosis. For example, there are scientific reports regarding pythiosis with haemoglobinopathies, anaemia and leukaemia as risk factors for infection in humans –12, and Phoca vitulina) and several species of cetaceans [Rhizopus sp. [Cryptococcus gatti [Candida albicans [Initial clinical examination and diagnostics implicated a fungal organism as a cause of disease in this case, although a specific identification at that time was not made. Marine mammals are known to be susceptible to fungal diseases. In particular, respiratory aspergillosis has been reported in harbour porpoises , harbouretaceans , 39. Raropus sp. , Cryptocus gatti  and Candalbicans  as causaDespite various antifungal and antibiotic treatments in this case, the clinical course subsequently developed into a progressive severe dermatitis. Retrospectively, such a clinical course, with difficulties in initial diagnosis and resistance to antifungal therapy, is typical for pythiosis, which is not a true fungal disease , 43, 44.P. flevoense as cause of this dermatitis was evidenced by its intralesional presence in combination with histological absence and microbiological exclusion of other potential pathogens, such as bacteria, true fungi, protozoa, and viruses. Also, the possibility of this particular Pythium species being an opportunistic infection was considered and excluded because: (1) there was no evidence from haematology, histopathology, or (molecular) microbiology to suggest underlying immunosuppression, and (2) infection occurred in a non-pregnant initially healthy young female harbour porpoise in good physical condition without co-morbidities. Furthermore, our findings of this new case of pythiosis in a harbour porpoise are consistent with observations from previous investigators that the host range of pythiosis is increasing [Pythium species, we recommend including P. flevoense as a differential diagnosis for dermatitis in marine mammals, and more specifically, in harbour porpoises that have been entangled in fishing nets.In conclusion, the diagnosis of ion, and  infectiocreasing , 5. So gAdditional file 1. Results of blood analyses for haematologic and biochemistry parameters from harbour porpoise Idun. Reference intervals from free-ranging harbour porpoises from Danish waters with a healthy clinical appearance  according to Siebert et al. [th and 90th percentiles for each blood parameter with their 95% bootstrapped confidence intervals, respectively. NA: not available.t et al. . The low


Human cytomegalovirus (HCMV) infection can lead to either lytic or latent infection, which is dependent on the regulation of the viral major immediate early promoter (MIEP). Suppression of the MIEP is a pre-requisite for latency and is driven by repressive epigenetic modifications at the MIEP during latent infection. However, other viral genes are expressed during latency and this is correlated with activatory epigenetic modifications at latent gene promoters. Yet the molecular basis of the differential regulation of latent and lytic gene expression by epigenetics is unclear. LUNA, a latent viral transcript, has been suggested to be important for HCMV latency and has also been shown to be important for efficient reactivation likely through its known deSUMOylase activity. Intriguingly, we and others have also observed that LUNA enhances latency-associated expression of the viral UL138 gene. Here, we show that in the absence of LUNA, the expression of multiple latency-associated transcripts is reduced during latent infection, which is correlated with a lack of activatory marks at their promoters. Interestingly, we also show that LUNA interacts with the hematopoietic transcription factor GATA-2, which has previously been shown to bind to a number of latency-associated gene promoters, and that this interaction is dependent on the deSUMOylase domain of LUNA. Finally, we show that the deSUMOylase activity of LUNA is required for the establishment and/or maintenance of an open chromatin configuration around latency-associated gene promoters. As such, LUNA plays a key role in efficient latency-associated viral gene expression and carriage of viral genome during latent carriage. Between 60 and 99% of global populations carry HCMV, depending on demographics; in part, this prevalence is made possible by the ability of the virus to establish lifelong persistence in the infected host. After primary lytic infection, HCMV can establish a quiescent or latent infection, which helps it avoid immune clearance . The lytDuring latent infection of undifferentiated myeloid cells, the MIEP is known to be targeted for repression to suppress the expression of key lytic IE genes by an overall balance of MIEP repressors ,7,8. HowAnother viral gene that has been shown to be expressed during latency is LUNA. It was one of the first genes identified to be transcribed during natural latency  and has In this manuscript, we further investigate this transcriptional phenotype and demonstrate that LUNA is important for the expression of multiple latency-associated transcripts, which correlates with increased levels of activatory epigenetic marks at latent promoters. In silico analyses reveal that multiple latent promoters encode binding sites for the hematopoietic transcription factor GATA-2, and interestingly, we show that LUNA interacts with transfected GATA-2 by co-immunoprecipitation (Co-IP). Finally, we link GATA-2 binding and the formation of an open chromatin conformation at latent viral promoters with the deSUMOylase motif of the LUNA protein. Together, these data argue that LUNA contributes to the regulation of expression of latency-associated HCMV gene expression in myeloid cells through an interaction with an important hematopoietic transcription factor, GATA-2.2 at 37 degrees. CD14+ cells were isolated from venous blood as described previously [THP1 cells were cultured in RPMI-1640 (Gibco) supplemented with 10% FCS and 5% penicillin/streptomycin in 5% COeviously ,26. TB40TB40E-SV40-GFP BAC was used to generate the LUNA mutant, LUNA knockout, and revertant viruses. For this, gBLOCKs from IDT  were utilised with primers for recombineering using the GalK/2-DOG selection method as described previously , using tTACCGCTTCGACGTCTTTGTCCGGTCAGGATCAGTGCCCGGGACAGTCCGCCTGTTGACAATTAATCATCGGCA 3′ GalK insert LUNA (primer) GGTCTCTTTCCACGGAGCAACGTCATGCGCGGCGCCGTCTCCGAGTTTCTTCAGCACTGTCCTGCTCCTT LUNA G81A stop (gBLOCK) TACCGCTTCGACGTCTTTGTCCGGTCAGGATCAGTGCCCGGGACAGTCCGGCTTGAGTGTCCGAGTCCTCGTCGCCGCTGGCCTCCTCGAAGCCGGCAAACATGGCTTCGGACAGGGGGGTCGGCGTCGGTGTGGATGAGAGGTCATCTTCGTCGTCCTCTTCCTCTTCTTCCTCCTCTTCCTCGGTGGGTGGTAATCCGGGGGACTGCGGGAGAAACTCGGAGACGGCGCCGCGCATGACGTTGCTCCGTGGAAAGAGACC LUNA G233C (gBLOCK)TACCGCTTCGACGTCTTTGTCCGGTCAGGATCAGTGCCCGGGACAGTCCGGCTTGGGTGTCCGAGTCCTCGTCGCCGCTGGCCTCCTCGAAGCCGGCAAACATGGCTTCGGACAGGGGGGTCGGCGTCGGTGTGGATGAGAGGTCATCTTCGTCGTCCTCTTCCTCTTCTTCCTCCTCTTCCTCGGTGGGTGGTAATCCGGGGGACTCCGGGAGAAACTCGGAGACGGCGCCGCGCATGACGTTGCTCCGTGGAAAGAGACC.LUNA C234G (gBLOCK) TACCGCTTCGACGTCTTTGTCCGGTCAGGATCAGTGCCCGGGACAGTCCGGCTTGGGTGTCCGAGTCCTCGTCGCCGCTGGCCTCCTCGAAGCCGGCAAACATGGCTTCGGACAGGGGGGTCGGCGTCGGTGTGGATGAGAGGTCATCTTCGTCGTCCTCTTCCTCTTCTTCCTCCTCTTCCTCGGTGGGTGGTAATCCGGGGGACTGGGGGAGAAACTCGGAGACGGCGCCGCGCATGACGTTGCTCCGTGGAAAGAGACC.5′ LUNA seq (60.4) (5′ primer for sequencing the mutants) GCG TGT TGC ACG CTC ACC 3′ LUNA seq (59.8) (3′ primer for sequencing the mutants) CCG CCG TGG GTT TTG GAC 5′ amp LUNA gB (55) TACCGCTTCGACGTCTTTG.3′ amp LUNA gB (55.5) GGTCTCTTTCCACGGAGC.® Green RT-qPCR kit according to the manufacturer’s instructions and the samples were amplified and detected using an ABI 7500 Fast Real-Time PCR machine (95 °C for 15 s and 60 °C for 45 s), as described previously [Chromatin immunoprecipitations were carried out as described previously . After sImmunoprecipitation analysis was carried out as described previously  using anIsopeptidase inhibitor, G5 (Sigma), was used as described previously . First, Quantification of viral and cellular mRNAs was carried out by SYBR green detection using primers described previously , except Genome copy number was determined using droplet digital PCR as described previously ,30. BrieTo investigate directly whether LUNA is important for latency-associated viral gene expression, we analysed the levels of UL138 and vIL-10 RNA during latent infection in the absence of LUNA. Recombinant viruses were generated in which a premature stop codon was engineered at the start of LUNA, resulting in viruses unable to express LUNA protein but which did not disrupt the UL82 gene on the complementary DNA strand (TB40E-LUNAmut), which was then controlled for through production of a revertant virus with LUNA repaired (TB40E-LUNArev). It is worth pointing out that this equivalent mutation of LUNA in the context of the Merlin clinical isolate of HCMV had little impact on the growth of Merlin in fibroblasts and the expression of viral gene products, including UL82 . The datTo further understand the mechanisms important for these differences in levels of UL138 and vIL-10 gene expression in the presence or absence of LUNA, we analysed the chromatin structure around the promoters of these latency-associated genes . ConsistWe previously identified that the hematopoietic transcription factor GATA-2, which plays an important role in host gene expression in myeloid cells, is also important for the expression of both LUNA and UL144 mRNAs during latency by GATA-2 binding to their promoters . IntriguThere is no evidence to suggest that LUNA can function as a general transcription factor; thus, we hypothesised that LUNA could regulate latent gene expression via interaction with GATA-2. To investigate whether LUNA might enhance expression from GATA-2-bearing promoters by a direct interaction between LUNA and GATA-2, we carried out interaction assays using co-immunoprecipitation in cells overexpressing GATA-2 and LUNA, which showed a clear interaction between GATA-2 and LUNA . FurtherWe observed that LUNA expression resulted in the formation of active chromatin on viral latency-associated promoters, that all these promoters contained GATA-2 transcription factor binding sites, and that LUNA interacted with GATA-2 in a deSUMOylase-dependent manner. Thus, we hypothesised that the chromatin signature observed with a LUNA deletion virus should be phenocopied with a LUNA deSUMOylase mutant. To test this, we generated a LUNA mutant virus in which we introduced a point mutation in the deSUMOylase domain of LUNA, rendering LUNA devoid of deSUMOylase activity (TB40E-LUNApoint), and tested whether the loss of the deSUMOylase activity from LUNA affected the transcription of viral genes. An analysis of infected cells shows that the expression of both vIL-10 and UL138 is reduced in cells latently infected with the TB40E LUNApoint compared to the control A,B. NextFinally, we wished to determine whether these LUNA-mediated changes could have any impact on the biology of latency. It is well established that HCMV infection extends the lifespan of classically short-lived CD14+ monocytes, which could be important for virus dissemination in vivo. Furthermore, we have observed that the expression of vIL-10 enhances the survival of infected CD34+ cells in long-term culture. Thus, we hypothesised that the defect in latency-associated gene expression observed in the absence of LUNA could have an impact on monocyte survival. To this end, CD14+ cells were infected with wild-type or a LUNA deletion virus and cultured for 10 days, and monocyte viability was assessed. The data show that viral infection improves cell survival compared to mock cells A, consisPrevious studies have demonstrated a clear role for the latency-associated gene product, LUNA, in latent carriage and reactivation from latency ,22. HereWhilst the absence of LUNA does not prevent the establishment of latency in CD14+ monocytes, we show here that it significantly enhances the expression of latency-associated genes, which we hypothesise is at least in part via the myeloid transcription factor GATA-2; this included the expression of vIL-10, which was significantly reduced in the absence of LUNA.We have previously shown that vIL-10 drives the expression of cellular IL-10 in monocytes ,31, and It is important to highlight that vIL-10 has pleiotropic roles in HCMV infection, with key roles in immune regulation. Thus, the enhancement of vIL-10 production by LUNA during latency could also be important for immune evasion during latency in vivo. For example, vIL-10 directly modulates the immune response  and the Our previous work has shown that LUNA is a functional deSUMOylase , and thiPML is generally repressive for viral IE gene expression, which would be advantageous during latency to help repress lytic gene expression. However, it is at present unclear how the virus maintains a repressive chromatin structure around the MIEP whilst at the same time allowing expression of latency-associated genes. The dispersal of repressive PML bodies during latency would be predicted to be disadvantageous for maintaining the general repressive chromatinization of the viral genome observed during HCMV latency; PML disruption is known to lead to activation of HCMV IE gene expression and gene expression of other viruses ,41. For In contrast, reactivation is likely regulated by the increase in differentiation-dependent transcriptional activators of the MIEP, coupled with a decrease in MIEP repressive factors, which can be enhanced by inflammatory signals that then allow viral IE gene expression  and progTaken together, we believe that the deSUMOylation function of LUNA not only ensures that reactivation from latency is primed to go, by removing repressive PML bodies in latently infected cells, but is also crucial for latent carriage by enhancing the transcription of latency-associated genes.


Nature Communications 10.1038/s41467-022-32395-w, published online 13 August 2022Correction to: The original version of this Article contained an error in Table 1. The correct version of the 3rd row (Circle part 2) of the 3rd column (Sequence (5’ -> 3’)) states ‘CGAGGTGCTTTTAGCACCTCGAAGTAAAGCTATCCACTGTCACCAACTACTAGATAAACGTCACACTTTTCGTGTGACG’instead of the original, incorrect ‘CGAGGTGCTTTTAGCACCTCGAAGTAAAGCTATCCACTGTCACCAACTACTAGATAAACGTCACACTTTTCGTGTGAC’ where the final ‘G’ was omitted.This has been corrected in both the PDF and HTML versions of the Article.