Staphylococcus aureus, Salmonella cholerae suis, Escherichia coli, and Streptococcus agalactiae. The MIC and MBC of additive A were the lowest for Staphylococcus aureus compared to that for the other three pathogens. The concentrations of 23 Chinese medicine ingredients -azulenone, 7-dihydroxy-2-(4-hydroxyphenyl)-8--6-chromen-4-one, acetylcholine, farrerol, pyrogallol, ethyl gallate, demethylwedelolactone, methyl gallate, kaempferide, gallic acid, eriodictyol, threonic acid, inositol, 3′,4′,7-trihydroxyflavanone, taxifolin, asiatic acid, and isorhamnetin) in additive A were significantly (p < 0.05 or p < 0.01) higher than those in additive B, respectively. It is concluded that the mixture composed of 23 active components in fermented compound Chinese medicine feed additive A plays an important role in inhibiting the growth of Staphylococcus aureus, Salmonella cholerae suis, Escherichia coli, and Streptococcus agalactiae.This experiment was conducted to compare the antibacterial ability and to identify the antibacterial components of different fermented compound Chinese medicine feed additives in order to develop one fermented compound Chinese medicine feed additive product that can effectively alleviate metritis, vaginitis, and mastitis of sows. The Oxford cup method and double dilution method were used to compare the antibacterial ability of three fermented compound Chinese medicine feed additives . UHPLC-QE-MS-based untargeted metabolomics was used to identify the antibacterial components of fermented compound Chinese medicine feed additives. Results showed that among fermented compound Chinese medicine feed additives A, B, and C, additive A had the strongest ability to inhibit the growth of  Escherichia coli, Staphylococcus aureus, Salmonella cholerae suis, and Streptococcus agalactiae , because an unstable implantation of fertilized eggs developed by the chronic uteritis and vaginitis of sows will increase embryo death, block fetal intrauterine development, or bring conception into failure . Milk pralactiae , 6, and alactiae –11. Suppalactiae –14, but alactiae . The solHouttuynia cordata, 23 parts of Folium artemisiae argyi, 40 parts of Thymifoious euphorbia herb, 8 parts of Cowherb seed, 25 parts of Potentilla discolor, 45 parts of Acalypha wilkesiana, 10 parts of sugar, 26 parts of wheat, 15 parts of soybean meal, 0.03 parts of Aspergillus niger, and 1.5 parts of yeast. Compound Chinese medicine feed additive B comprised 20 parts of Houttuynia cordata, 15 parts of Folium artemisiae argyi, 40 parts of Thymifoious euphorbia herb, 25 parts of Potentilla discolor, 45 parts of Motherwort, 14 parts of Licorice, 18 parts of sugar, 8 parts of wheat, 8 parts of soybean meal, 0.05 parts of Aspergillus niger, and 1.0 parts of yeast. Compound Chinese medicine feed additive C comprised 25 parts of Folium artemisiae argyi, 12 parts of Cowherb seed, 30 parts of Potentilla discolor, 25 parts of Thymifoious euphorbia herb, 30 parts of Acalypha wilkesiana, 25 parts of sugar, 10 parts of soybean meal, 0.02 parts of Aspergillus niger, and 0.7 parts of yeast. Traditional Chinese medicines, wheat and soybean meal were ground into powder using a pulverizer to pass through 80-mesh sieves.According to the components and pharmacological characteristics of traditional Chinese medicines, the following traditional Chinese medicines were selected to compose three compound Chinese medicine feed additives. Compound Chinese medicine feed additive A comprised 20 parts of Aspergillus niger and yeast were activated in this sugar solution for 30 min to produce a kind of fermentation liquid. The compound Chinese medicine feed additive was mixed with fermentation liquid, packed with plastic bags, and put into an oven for fermentation at 33°C for 36 h. The fermented additives were taken out and packed by vacuum sealing and stored at room temperature for 15 days.Tap water was firstly weighted at a ratio of 1:1 according to the weight of compound Chinese medicine feed additive and heated to 40°C to dissolve sugar, then Fifty gram of each fermented compound traditional Chinese medicine feed additive was soaked with 500 ml distilled water in a beaker for 1 h; then, the liquid mixture in the beaker was heated to boil and kept boiling for 1 h and filtered with 8 layers of gauze. The filter liquor was transferred into a glass cup, and the filter residue was washed into the beaker and boiled with 300 ml distilled water for 1 h; later, the mixture was filtered again. The twice-filtered liquors were mixed together and concentrated to 25 ml in an oven at 65°C with a final concentration of 2 g/ml; the concentrated liquor was sterilized by high-pressure steam and stored at −20°C for subsequent tests.Streptococcus agalactiae (GDMCC 1.768), Escherichia coli (GDMCC 1.176), Staphylococcus aureus (GDMCC 1.174), and Salmonella cholerae suis (GDMCC 1.163) were purchased from Guangdong Microbial Culture Collection Center. 0.3 ml brain heart infusion (BHI) broth was pipetted into the lyophilized tube containing Streptococcus agalactiae, then the tube was vortexed gently until the strain powder was completely dissolved; the suspension was pipetted into 5 ml BHI broth and cultured in an incubator at 37°C for 48 h. 0.3 ml Lennox broth (LB) was pipetted into the lyophilized tubes containing Escherichia coli, Staphylococcus aureus, and Salmonella cholerae suis; respectively, all tubes were vortexed gently until the strain powder was completely dissolved, then the suspension was respectively pipetted into 5 ml LB broth and cultured in an incubator at 37°C for 24 h. The optical density (OD) value of bacterial solution was measured at 660 nm to reach a final concentration of 1.5 × 108 CFU/ml.Escherichia coli suspension  was uniformly spread on the surface of agar and three Oxford cups (7.8 * 6 * 10 mm) marked A, B, and C were placed on the agar medium in each petri dish. Two-hundred microliter of concentrated liquor A, B, or C was added to the corresponding Oxford cup; each strain had 3 replicates. All petri dishes were incubated at 37°C for 18 h and then taken out for the measurement of diameter of bacteriostatic circles. The blood culture medium was prepared to measure the abilities of concentrated liquor A, B, and C against the growth of Streptococcus agalactiae with the above method. Among the three concentrated liquors, the one with the best bacteriostatic effects against Streptococcus agalactiae, Escherichia coli, Staphylococcus aureus, and Salmonella cholerae suis was selected to determine its minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC).The tryptone soybean agar (TSA) was poured into the petri dishes; after agar solidification, a total of 0.1 μl Escherichia coli, Staphylococcus aureus, and Salmonella cholerae suis) or with BHI broth  to give concentrations ranging from 1 to 0.0078125 g/ml in 8 culture tubes, and the ninth tube was only added with 2 ml LB broth as the positive control. Fifty microliter of bacteria solution was added into each tube, and all tubes were incubated for 18 h at 37°C. The lowest concentration of the selected concentrated liquor that caused a visible inhibitory effect was defined as the MIC. The MBC was determined by incubating 10-μl samples from each tube with an inhibitory effect on plates with TSA  or with blood culture medium  at 37°C for 18 h, with the lowest concentration of sample with no visible sign of growth determined as the MBC. All experiments were conducted in three replicates.The double dilution method was used to determine the MIC. The selected concentrated liquor was successively diluted with LB broth  coupled with Q Exactive focus MS/MS . Chromatographic separations were performed using a Waters ACQUITY system equipped with an ACQUITY UPLC BEH C18 column . The mobile phase consisted of 0.1% formic acid water (A) and 0.1% formic acid acetonitrile (B) at the flow rate of 0.5 ml/min, and the injection volume was 4 ml. The gradient program was as follows: 85–25% B (0–11.0 min), 25–2% B (11.0–12.0 min), 2–2% B (12.0–14.0 min), 2–85% B (14.0–14.1 min), 85–85% B (14.1–15.0 min), and 85–85% B (15.0–16.0 min). The ESI source was applied to analyze the chemical composition in both positive and negative ion modes with full scan/ddMS2. The MS parameters were set as follows: the scan range was 50–1,000 m/z, the spray voltages were set at 4.0 and 3.6 kV in positive and negative modes, respectively, sheath gas was 35 arb, auxiliary gas was 10 arb, the capillary temperature was 400°C, the maximum injection time for MS1 and ddMS2 was 100 and 45 ms, respectively, and the resolutions for MS1 and ddMS2 were 70,000 and 17,500, respectively; putative molecules of interest were fragmented using three different collision energies .p < 0.05 were selected as the differential variables; metabolite identification was performed by searching the in-house database and web databases .Raw data of UHPLC-QE-MS were processed using the XCMS online platform, and the qualified data were uploaded to SIMCA-P  for multivariate statistical analysis. Partial least-square-discriminate analysis (PLS-DA) and orthogonal partial least-square-discriminate analysis (OPLS-DA) were performed to find out the metabolic distinction. The variable importance of projection (VIP) values was used to characterize the contributions of the metabolites to the change rates of Y variety in OPLS-DA, and metabolites that meet these criteria including MS2 score > 0.80, VIP > 1.0, and To further investigate the mechanism of targeted differential metabolites, the network pharmacology approach was introduced to uncover the related pathways, compounds, enzymes, and reactions. Compounds with MS2 score > 0.80, VIP > 1.0, fold change > 1, and o <0.05 were selected as the target differential metabolites and used to construct the network with FELLA R package . The nodt-test; the value at p < 0.05 was considered as significance threshold, and the results were presented as mean ± SE.Statistical analysis was performed by SPSS 17.0 , comparisons between two groups were made with Student's Streptococcus agalactiae, Escherichia coli, Staphylococcus aureus, and Salmonella cholerae suis were different and ranked as follows: A > C > B. Results in Staphylococcus aureus but had the worst effect in preventing the growth of Streptococcus agalactiae, additive B had no bacteriostatic effect, and additive C had the ability in controlling the growth of Streptococcus agalactiae, Staphylococcus aureus, and Salmonella cholerae suis but had no bacteriostatic effect on Escherichia coli.The abilities of additives A, B, and C in inhibiting the growth of Staphylococcus aureus among all tested bacteria.Fermented compound Chinese medicine feed additive A was used to determine MIC and MBC because additive A had the best bacteriostatic effect compared to the other two additives, and data in p < 0.05 were selected as the target compounds. Results in In order to find out why additive A had better antibacterial ability than additive B, the metabolomics untargeted analysis was performed to identify the differential metabolites between additives A and B, and metabolites with MS2 score > 0.80, VIP > 1.0, fold change > 1, and via the related enzymes and reactions: D-glucose, ascorbate, choline, D-ribose, oxalate, deoxyguanosine, xanthine, guanosine, sinapate, alpha-D-ribose 1-phosphate, 2-deoxy-D-ribose 1-phosphate, 1-O-sinapoyl-beta-D-glucose, 3-dehydro-L-threonate, kaempferol, homoeriodictyol, and eriodictyol chalcone.Network analysis was conducted in order to reveal the molecular network and mechanism of the interaction of the KEGG pathways with related compounds, enzymes, modules, and reactions. Twenty-three differential compounds were mapped with the KEGG database, and only five compounds  indicated as green square in Staphylococcus aureus, Salmonella cholerae suis, Escherichia coli, Streptococcus, and Bacillus cereus -azulenone, 7-dihydroxy-2-(4-hydroxyp-henyl)-8--6-chromen-4-one, acetylcholine, farrerol, pyrogallol, ethyl gallate, demethylwedelolactone, methyl gallate, kaempferide, gallic acid, eriodictyol, threonic acid, inositol, 3′,4′,7-trihydroxyflavanone, taxifolin, asiatic acid, and isorhamnetin than fermented compound Chinese medicine feed additive B; most of them and their derivatives have strong bacteriostatic and/or bactericidal effects against pathogens, and this is the reason why fermented compound Chinese medicine feed additive A has better bacteriostatic and/or bactericidal effects than fermented compound Chinese medicine feed additive B.Staphylococcus aureus, Salmonella cholerae suis, Escherichia coli, and Streptococcus agalactiae; the mixture composed of 23 active components in fermented compound Chinese medicine feed additive A exerted bacteriostatic and/or bactericidal effects on the tested pathogens. The sensitivity of these four bacteria to fermented compound traditional Chinese medicine feed additive A is ranked as Staphylococcus aureus > Salmonella cholerae suis > Escherichia coli > Streptococcus agalactiae.Additive A had the strongest ability to inhibit the growth of The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding authors.YH and WL: conception, experimental design, and manuscript preparation. WZ, HH, and YC: investigation. WZ and HW: data analysis. All authors have read and agreed to the published version of the manuscript.This work was supported by the Key Research and Development Plan of Jiangxi Province (20192BBFL60021).Jiangxi Jiabo Bioengineering Co., Ltd., was affiliated with this study. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The large amount of knowledge regarding epigenetic pathways has opened a broad range of treatments that provide hope for adult patients with highly aggressive forms of solid tumors. The most commonly used treatments for epigenic modifications are based on the specific inhibitors of DNA methyltransferases, azacitidine and decitabine (5-AZA-dC), and on histone deacetylases inhibitors, such as trichostatin A (TSA) or vorinostat (SAHA). However, many other compounds are under investigation, and some are being evaluated in clinical trials. In this review, we have extracted relevant information about epigenetic pathways and treatments that target epigenetic modifications in highly aggressive tumors, as a new hope for these patients.Highly aggressive tumors are characterized by a highly invasive phenotype, and they display chemoresistance. Furthermore, some of the tumors lack expression of biomarkers for target therapies. This is the case of small-cell lung cancer, triple-negative breast cancer, pancreatic ductal adenocarcinoma, glioblastoma, metastatic melanoma, and advanced ovarian cancer. Unfortunately, these patients show a low survival rate and most of the available drugs are ineffective. In this context, epigenetic modifications have emerged to provide the causes and potential treatments for such types of tumors. Methylation and hydroxymethylation of DNA, and histone modifications, are the most common targets of epigenetic therapy, to influence gene expression without altering the DNA sequence. These modifications could impact both oncogenes and tumor suppressor factors, which influence several molecular pathways such as epithelial-to-mesenchymal transition, WNT/β–catenin, PI3K–mTOR, MAPK, or mismatch repair machinery. However, epigenetic changes are inducible and reversible events that could be influenced by some environmental conditions, such as UV exposure, smoking habit, or diet. Changes in DNA methylation status and/or histone modification, such as acetylation, methylation or phosphorylation, among others, are the most important targets for epigenetic cancer therapy. Therefore, the present review aims to compile the basic information of epigenetic modifications, pathways and factors, and provide a rationale for the research and treatment of highly aggressive tumors with epigenetic drugs. DNA is organized inside the nucleus, in a very complex structure called chromatin. The negative charge of DNA is supported by basic proteins that are rich in arginine and lysine residues, called histones. There are five families of histones and according to their function they are called core histones H2, H3, and H4) that form the nucleosome core, or linker histones H1 and H5), which contribute to the condensation of the nucleosome. The nucleosome core is composed by two H2A–H2B dimers and a H3–H4 tetramer. The electrostatic attraction between the positively charged histones and negatively charged DNA allows the complex structure of chromatin to form . Th. Th39]. On the other hand, readers of these modifications determine the functional outcome of specific epigenetic change. Some of the proteins involved in the recognition of histone modifications are BET . This family is composed of four proteins , and plays important roles in tumor development, since they also lead to transcriptional activity ,51. For This family includes several factors, but the most notable, in regards to cancer, are small interfering RNA (siRNAs), microRNAs (miRNAs), PIWI-interacting RNA (piRNAs), and long non-coding RNAs (lncRNAs)  5]..5].The small interfering RNA (siRNA) transcripts are double-stranded RNA fragments, about 21–25 base pairs long. The function of siRNA is thought to be related to erasing viral double-stranded sequences to avoid infection. SiRNA is cleaved by Dicer from long double-stranded RNA sequences . The douMiRNA are the most known non-coding RNA and they are involved in several cell functions. Several miRNAs are linked to cancer initiation and development. Furthermore, miRNAs can be tumor-prone or tumor-suppressive factors . MiRNAs P-element-induced wimpy testis (PIWI) proteins belong to the Argonaute (AGO) family and were discovered in the germline . They alLong non-coding RNAs (lncRNAs) constitute a huge subgroup of ncRNAs, defined as RNA transcripts, with more than 200 nucleotides . LncRNAsThe large amount of knowledge regarding epigenetic modifications has opened a broad palette of treatment strategies for the most aggressive solid tumors in adulthood. Thus, the objective of this review is to compile basic knowledge about epigenetic pathways and treatments, and provide a rational for further clinical trials, based on the use of these treatments in highly aggressive solid tumors.The most commonly drugs used as hypomethylating agents are specific inhibitors of DNA methyltransferase (DNMT), for example, azacitidine and decitabine (5-AZA-dC)  70]. Th. Th70]. TRIM36 hypermethylation, and its subsequent inhibition is associated with the acquisition of an aggressive phenotype [Small-cell lung cancer (SCLC) incidence over time has decreased, reducing by 10–11% in all the cases of lung cancer, which may reflect decreases in smoking habits and changes in the type of cigarettes . One of henotype . SCLC ishenotype .P16, TERT, WT1, and RASSF1), which could improve the efficiency of SCLC diagnosis when compared with cytologic evaluation [DCLK1, which has been associated to colorectal cancer and cholangiocarcinoma, has been found in liquid biopsies in 75% of SCLC patients, and has been associated with poor survival; therefore, this could represent a promising biomarker for early diagnosis and disease prognostic for this cancer subtype [ITK, RUNX3, CTLA4, PLG, EMR3, SLC22A18, TRIP6IL10, PECAM1, S100A2, MMP9, ERCC1, CSF3R and CAV1 [The methylation status of bronchial washings from different types of lung cancers provided a signature, based on four DNA methylated factors  comprise 7–14% of all breast cancers . TNBC isBRCA1, which implies a deficient DNA repair machinery. Genome-wide DNA methylation analysis in TNBC supports that hypermethylation causes the downregulation of PRSS8, VAMP8 and CLDN4 factors, which confer mesenchymal features [BRCA1 methylation in a TNBC basal-like subtype. This finding could imply resistance to PARP inhibitors for the treatment of BRCA-mutant basal-like TNBC [TP53, one study has demonstrated that the use of zinc metallochaperones (ZMCs) is efficient to reactivate zinc-deficient mutant TP53, by restoring its zinc binding. The use of ZMC1 with a mutation in R175HTP53 restores TP53 reactivation [PDLIM4 or VHL, through demethylation, and showed a high anti-proliferative effect in TNBC models [A high percentage of TNBC patients carry germline/somatic mutations or epigenetic silencing in features . One stuike TNBC . As mosttivation . Anothertivation . CD24 ovtivation . MutatioC models .TGFβR2 promoter in the TNBC cell line, MDA-MB-231. Moreover, the inhibition of TGFβR2 decreased migration of the cell line [Concerning histone acetylation, one study showed high levels of H3K9 acetylation in the ell line . Anotherell line . EZH2 woell line .In respect to ncRNA, the presence of hypermethylation at miR-31 loci in TNBC has been described. Moreover, miR-31 maps to the sequence of a novel long non-coding RNA, LAOT554202 . Both arBRCA1 or BRCA2 , BRCA1 (45.8%), P16/INK4a (35.4%), P15/INK4b (35.4%), RARβ (35.4%), and P73 promoters (33.3%) in PDAC patients. Moreover, other genes are methylated to impair several signaling pathways, such as TGF-β, WNT, integrin or ROBO [Pancreatic ductal adenocarcinoma (PDAC) shows the lowest five-year survival rate, around 3%, and it is the fourth leading cause of cancer-related deaths in men and women . It is opatients . Furthertability . A recenand MLL2 . In addiand MLL2 . Both hiand MLL2 . Increasand MLL2 . In fact or ROBO .P300 supports its role as a tumor-suppressive gene in PDAC [Concerning histone-modifying enzymes, aberrant HATs and HDACs have been found in PDAC. One study, performed in PDAC-derived cell lines, showed an inhibition of the expression of HAT, P300, and a secondary upregulation of several miRNAs . The sup in PDAC . The abe in PDAC ,126. Als in PDAC . Other s in PDAC . Histone in PDAC . The HAT in PDAC .Concerning miRNA, one study with PDAC patients revealed a poor prognosis signature based on the deregulation of 64 miRNAs, and the upregulation of miR-21, miR-196a-2, miR-203, miR-155, miR-210, and miR-222 . FurtherG12DKRAS to promote tumor initiation in in vivo models [MUC2 promoter region regulates MUC2 gene expression, and this expression could be positively modulated by treatment with trichostatin A (TSA) and 5-aza [The impact of bromodomain inhibitors has also been evaluated in PDAC. BRD4770 is an inhibitor of G9a that induces PDAC autophagy . Moreoveo models . It has nd 5-aza . Anothernd 5-aza . Anothernd 5-aza . Interlend 5-aza .In clinical trials, a pilot study with relapsed patients  is the most commonly diagnosed tumor in elderly Caucasian men . UnfortuMGMT (O-6-methylguanine DNA methyltransferase) hypermethylation predicts BCNU (carmustine) and temozolomide response in gliomas [ gliomas ,157. Mor gliomas . Treatme gliomas . Some HD gliomas .Recently, it has been described that a specific GBM subtype, with high levels of MGMT, expresses methyl-CpG binding domain 3 (MBD3) protein, which targets CK1A. Therefore, this subtype of patients may obtain benefit from CK1A activator pyrvinium pamoate (Pyr-Pam), leading to MBD3 degradation . The newConcerning clinical trials, the use of temsirolimus has obtained interesting improvement in 36% of treated patients; furthermore, the treatment achieved a significantly longer time to progress . In contIn conclusion, although molecular diagnosis has brought new options to identify and treat patients, therapeutic options remain without any significant changes. Currently, the best standard treatment is the maximum safe resection, followed by chemoradiation and adjuvant chemotherapy. We hope that new clinical trials with epigenetic target therapies could improve the responses to conventional treatments.CIP1/P21 or P16/INK4a [CCR7) and CXC chemokine receptor 4 (CXCR4) are epigenetically upregulated in melanoma cells, and have the ability to induce metastasis of melanoma [DAPK), O6-methylguanine DNA methyl-transferase (MGMT), RAS association domain family protein 1A (RASSF1A), and retinoic acid receptor-β2 (RAR-β2). The hypermethylation of DAPK, MGMT and RASSF1A is significantly lower in the early stages than in the advanced stages, whereas the incidence of hypermethylation of RAR-β2 is highly similar in the early and advanced stages [RAR-β2 methylation in melanoma cells, and it achieved the highest benefits in combination with retinoids [BRAFV600E and PTEN promote malignant transformation of melanocytes [CDKN1A expression and arrest cell proliferation [The main issue with metastatic melanoma lies in its chemoresistance. Currently, the new immunecheckpoint inhibitors against CTLA-4, PD-1 or PD-L1 have improved patient outcome. However, secondary genomic aberrations make tumor cells acquire rapid resistance to these therapies . One of 16/INK4a . The epi16/INK4a . CC chemmelanoma . The fold stages . The HDAetinoids . Also, Tetinoids . It has anocytes . EZH2 isferation .3, a potential therapy for metastatic melanoma [Concerning ncRNA, several studies have reported the importance of miRNA regulation in melanoma. For example, miRNA-125b is involved in the regulation of vitamin D receptor (VDR), and in the resistance of 1,25-dihydroxyvitamin Dmelanoma . Moreovemelanoma .ARF promoter, and upregulate its expression [INK4A promoter, leading to its subsequent activation [Since the largest clinical issue in the treatment of advanced melanoma patients is chemoresistance, the effort of researchers is centered around the discovery of a new treatment method to improve drug sensitivity. Interleukin-2 has exhibited potent antitumor activity in the fight against melanoma; nevertheless, its high toxicity has limited its use . Treatmepression . Treatmetivation . Anothertivation . Some istivation . Immune tivation . Perhapstivation .Clinical trials with epigenetic therapy in metastatic melanoma have been mostly based on decitabine and other epigenetic modulating drugs, such as histone deacetylase inhibitors. A phase I clinical trial has explored the safety and tolerability of two epigenetic drugs, decitabine and panobinostat (a histone deacetylase inhibitor), in combination with temozolomide, to overcome chemoresistance in advanced melanoma  are the gynecological cancers with the highest mortality rate, probably because most AOT patients present advanced stages at diagnosis (stage III or IV), due to the lack of symptoms or unavailable specific screening biomarkers . While r I HDACs . Also, c I HDACs . SIRT1 utylation . SIRT3, tylation . Other ftylation . Furthertylation . Anotherpatients . Anotherpatients , and thepatients ,215. Othpatients .It is known that cancer modifies the microenvironment to inhibit the immune system. In this context, the overexpression of HLA-class I and II has been associated to AOT . EpigeneThe inhibition of histone acetyltransferase is a new approach for the treatment of malignant AOT and its chemoresistance. The following three HDAC inhibitors have been approved by the FDA: romidepsin, panobinostat, and vorinostat . TrichosAOC is strongly influenced by epigenetic changes that affected DNA methylation and histone modifications. The first attempts to modify the epigenetic of AOC with drugs have achieved low response rates as single agents; thus, their combination with targeted therapies, based on the mutational burden of tumors, must be evaluated.IDH1/2, EZH2 or DNMT3A. Moreover, not all mutations are tumor-prone, and we must consider tumor-suppressive factors such as KDM6A and CREBBP/P300 [In the clinic, patients with highly aggressive tumors are presented with different prognoses, despite having a similar stage and grade of cancer. These observations could be explained by the tumor heterogeneity that is characterized by several epigenetic modification profiles . FirstlyBBP/P300 . FinallyBBP/P300 . All the


Lithobiomorpha, with only 100 species and subspecies known from the country. Altogether, 11 species of subgenusMonotarsobius have been recorded from China, but only two of them have been reported from Hebei Province. Herein, a new species recently discovered in the Hebei Province, China, is described and illustrated.The myriapod fauna of China is still poorly known and very little attention has been paid to the study of Lithobius (Monotarsobius) tetrasulcus sp. n. is described and illustrated from Hengshui Lake National Nature Reserve, Hebei Province, China. The new species is compared with Lithobius (Monotarsobius) crassipes Koch, 1862 from Taiwan, China. It can be easily distinguished from congeners by having a longitudinal groove on the dorsal side of the femur and tibia of the male legs 14 and 15, only having a posterior spine on the dorsal side of femur of legs 12–15, lacking robust spines lying dorsally on the external margin on the second article of the female gonopods and the third article of the female gonopods having a bidentate apical claw.A new lithobiids species  Monotarsobius was originally proposed as a subgenus of Lithobius Leach, 1814 in the family Lithobiidae by Verhoeff . The terminology of the external anatomy follows Specimens were collected under leaf litter and/or stones and preserved in 75% ethanol. Illustrations and measurements were produced using a ZEISS SteREO Discovery.V20 microscope equipped with an Abbe drawing tube and an ocular micrometre and Axiocam 512 colour. The colour description is based on specimens fixed in 75% ethanol. The body length is measured from the anterior margin of the cephalic plate to the posterior end of the postpedal tergite. Type specimens and other material are deposited in the School of Life Sciences, Hengshui University, Hengshui, China  oval to rounded ocelli on each side, from small to large, arranged in three irregular rows, the posterior ocellus largest. Ventral ocelli slightly smaller than the dorsal, domed, translucent and usually darkly pigmented Fig. B.Tömösváry’s organ Fig. B, To cloForcipular coxosternite subtrapezoidal Fig. D, anteriTergites smooth, without wrinkles, dorsum slightly convex; tiny setae emerging from pores scattered sparsely over the entire surface, near the margin with few long setae; T1 narrower postero-laterally than antero-laterally, generally inverted trapezoidal; TT1 and TT3 narrower than the cephalic plate, T3 wider than the T1. Lateral marginal ridges of all tergites continuous. Posterior margin of TT1, 3 and 5 continuous, posterior margin of TT8, 10, 12 and 14 discontinuous; posterior margin of T7 straight, posterior marginal ridges of TT3, 5, 8, 10, 12 and 14 with concavity gradually increasing. Posterior angles of tergites rounded, without triangular projections. Miniscule setae scattered sparsely over the surface, one thick and long setae on both anterior angles of each tergite.Sternites. Posterior sides of sternites narrower than anterior, generally inverted trapezoidal, smooth; setae emerging from sparsely scattered pores on the surface and lateral margins, very few short setae sparsely scattered amongst them. Short and thick setae on the surface of the anterior part slightly increasing from S11 to S15.Legs relatively robust, tarsi articulation ill-defined on legs 1–13, well-defined on legs 14 and 15. All legs with moderately long curved claws; legs 1–13 with anterior and posterior accessory spurs, anterior accessory spurs moderately long and slender, forming a moderately small angle with the claw, posterior accessory spurs slightly more robust, forming a comparatively large angle with the claw, legs 13 with anterior accessory spurs; anterior accessory spurs of legs 14 short and thick, posterior accessory spurs normal; legs 15 lacking accessory spurs. From short to long setae sparsely scattered over the surface of coxas, trochanters, prefemora, femora and tibiae of all legs, more setae on the tarsal surface; setae on the dorsal and ventral surfaces slightly longer than the anterior and posterior ones; legs 14 and 15 thicker than the anterior pairs in both female and male, legs 15 thicker and stronger in male than in female. Many glandular pores on the posterior surfaces of legs 14 and 15. Tarsus 2 is 3.5–4.4 times longer than wide, tarsus 2, 46.0%–69.1% length of tarsus 1 on legs 15 in female; tarsus 2, 3.9–4.9 times longer than wide, tarsus 2, 60%–70% length of tarsus 1 on legs 15 in male. Leg plectrotaxy as in Table Coxal pores 3–6 in a row, 4-5(6)-5-4, 3(4)-5-5(4)-3(4), 4-4(5)-4-4, commonly 4-5-5-4 in female, 3-4(5)-4-3(4), commonly 3-4-4-3 in male; slightly oval or round, coxal pore field set in a relatively shallow groove, the coxal pore-field fringe with slight prominence, with moderately long setae sparsely scattered over the surface.Female. S15 anterior margin broader than posterior, generally inverted trapezoidal, postero-medially straight. Moderately long setae sparsely scattered on S15 surface. Surface of the lateral sternal margin of genital segment well chitinised, posterior margin of genital sternite deeply concave between condyles of gonopods, except for a small, median rhombic-shaped bulge. Relatively long setae very sparsely scattered over ventral surface of the genital segment, slightly more setae on posterior part, especially on the posterior edge. Gonopods: first article fairly broad, bearing 16 or 17 moderately long setae, arranged in three irregular rows; with 2+2 small coniform spurs, inner spur slightly smaller than the outer  crassipes Koch, 1862 (L. (M.) crassipes; with posterior spine on the dorsal of femur of legs 12–15, other than legs 10–14 in L. (M.) crassipes; lacking robust spines lying dorsally on the external margin on the second article of the female gonopods in contrast to the presence of three robust spines in L. (M.) crassipes; lacking robust spines lying dorsally on the external margin on the third article of the female gonopods in contrast to the presence of one robust spine in L. (M.) crassipes.The new species is morphologically close to ch, 1862  from Tai


RRM2B plays a crucial role in DNA replication, repair and oxidative stress. While germline RRM2B mutations have been implicated in mitochondrial disorders, its relevance to cancer has not been established. Here, using TCGA studies, we investigated RRM2B alterations in cancer. We found that RRM2B is highly amplified in multiple tumor types, particularly in MYC-amplified tumors, and is associated with increased RRM2B mRNA expression. We also observed that the chromosomal region 8q22.3–8q24, is amplified in multiple tumors, and includes RRM2B, MYC along with several other cancer-associated genes. An analysis of genes within this 8q-amplicon showed that cancers that have both RRM2B-amplified along with MYC have a distinct pattern of amplification compared to cancers that are unaltered or those that have amplifications in RRM2B or MYC only. Investigation of curated biological interactions revealed that gene products of the amplified 8q22.3–8q24 region have important roles in DNA repair, DNA damage response, oxygen sensing, and apoptosis pathways and interact functionally. Notably, RRM2B-amplified cancers are characterized by mutation signatures of defective DNA repair and oxidative stress, and at least RRM2B-amplified breast cancers are associated with poor clinical outcome. These data suggest alterations in RR2MB and possibly the interacting 8q-proteins could have a profound effect on regulatory pathways such as DNA repair and cellular survival, highlighting therapeutic opportunities in these cancers. RRM2B encodes a small subunit of p53-inducible ribonucleotide reductase (RNR). The RNR is a heterotetrametric enzyme responsible for the de novo conversion of ribonucleotide diphosphates into the corresponding deoxyribonucleotide diphosphates for DNA synthesis, thus playing an important role in maintaining deoxyribonucleotide pools , with distinct but variable clinical phenotype  (amplifications and deletions) have decreased overall survival (OS)  . Similark cancer , esophagk cancer  and lungk cancer . Anotherl cancer .RRM2B amplification may be driven by selection of RRM2B function, or RRM2B may be amplified as a passenger, concurrent with selection for a nearby gene with a driver activity in cancer. In breast cancer, multiple genes localized in the 8q12.1–8q24.23 interval were found to be amplified, including RRM2B , but also exhibit distinct mutation signatures. Analysis of the most common breast cancer subtype indicated that RRM2B amplifications may independently impact clinical outcomes in these cancers. Further, tumors bearing RRM2B amplifications showed that several genes in the 8q22–8q24 region  are highly amplified. Additionally, analysis of 8q-proteins suggests functional interaction within the same cell regulatory mechanisms of DNA damage response and repair, hypoxia and apoptosis. Based on these results, we hypothesize that while MYC may be the cancer driver, co-amplification of RRM2B and other 8q-genes may be relevant for cancer cell survival. These finding suggest opportunities for novel therapeutic targeting strategies (such as those targeting DNA damage response and repair) for tumors carrying RRM2B alterations.Here, using data from TCGA, we found that 2 . The truncating mutations in TCGA are frameshift deletions, frameshift insertions, nonsense, splice site. All mutations were analyzed for co-occurrence, including those that were homozygous or heterozygous.TCGA studies were accessed using the cBioPortal website2 . Data waRRM2B there was only a single case of deep deletion out of all , observed in BRCA, and was removed. For CCNE2, EI3FE, MTDH, MYC, RAD21, TP53INP1, and YWHAZ—there were no cases of deep deletion for any of the genes in the HNSC study, but there was one case each in EI3FE, RAD21 and YWHAZ with deep deletions in the BRCA study, and also one case each in EI3FE and MTDH as well as two cases in RAD21 for the OV study. V2 RSEM from cases with different copy number variants categories and statistical significance was tested in Graphpad Prism 8.0 using Mann-Whitney Non-Parametric T-test , BRCA (breast cancer study) and HNSC (head and neck cancer study) studies on 11/7/2018, and 3/19/2020. The RNA seq. data were only analyzed for tumors that were also profiled for copy number variants. Cases were segregated based on copy number variants type: deep deletion, shallow deletion, diploid, gain and amplification. For c T-test . Additioc T-test  and log c T-test  p-values3 on 11/5/2018. Next, IDs of subjects with different cancer types were downloaded from the cBioPortal website2, and subjects with amplification in RRM2B and MYC genes were selected. Presence or absence of these amplifications classified the subjects into four categories: (1) cases with amplification in both RRM2B and MYC, (2) cases with amplification in RRM2B, but not in MYC, (3) cases with amplification in MYC, but not in RRM2B, (4) and cases without amplification in both genes. For each specific cancer type, mutational spectra of each category were calculated by finding the average number of amplifications in each of the 96 mutation classes. To compare differences in these 96 mutation types, one-way ANOVA  cases with amplification in RRM2B, but not in MYC, (3) cases with amplification in MYC, but not in RRM2B, (4) and cases without amplification in both genes, and anova1, anova2 and Wilcoxon rank-sum functions in MATLAB R2018a were used to test for significance. Each signature type was determined to be significant only if its P-value was below 0.05 in the one-way ANOVA test. The two-way ANOVA was used to distinguish the impact of factors such as co-amplifications with MYC. Additionally, the Wilcoxon rank-sum test was also performed as a secondary method to test for significance.Tumor whole-exome sequence data in the TCGA Pan-Cancer Atlas studies , that includes spectra of individual tumors, was downloaded from the Synapse platformay ANOVA , two-wayay ANOVA , and Wilay ANOVA  were useay ANOVA  was applRRM2B or MYC, in both or neither. The clinical data from each group were used to generate OS and disease/progression-free survival (DFS) Kaplan-Meier curves  TCGA study. Patients were divided into different groups based on having amplifications in r curves  using Grr curves  and signlso used , and sigcbioportal.org. The genes used for analysis are located on chromosome 8 from the region 8q11.2–8q24.3 and have been associated with cancer according to the Cancer Index4. The amplification pattern of other 8q-genes in BC, OC, and HNSCC was compared to cases with co-amplifications of MYC and RRM2B, cases with single amplifications or cases that were unaltered. A univariate analysis was conducted to calculate the positive or negative linear trend for the frequency of gene amplification, assessed at 95% confidence interval. The Pearson correlation coefficient and p-values were computed by using PROC CORR in SAS 9.4. The significance level was set at p < 0.05.The tumors that were assessed for alteration frequency were also analyzed for amplifications in the 8q-genes. Tumor data was downloaded on 6/22/18 from p-values, and FDR thresholds at 0.01.Data for the RRM2B interaction network was downloaded from the BioGRID database  10/25/2. SeveralRRM2B is frequently amplified, with the highest percentage observed in ovarian, breast, bladder, and liver cancers (21.54–14.5%), and a lower rate of amplifications in multiple other cancers (14–0.6%) (RRM2B were only observed in Non-Hodgkin’s lymphoma (∼2%). In addition to amplifications, a low frequency of mutations (<2%) were observed in head and neck, lung, endometrial, esophagogastric, cervical, and pancreatic cancers  . Deletio cancers . Mapping/regions , unlike /regions . Only th/regions . This muMYC  did not have RRM2B amplifications  and head and neck squamous cell carcinoma (HNSCC), this was half of all cases with MYC amplifications, while in breast cancer (BC) this was ∼90% cases   .RRM2B and/or MYC were amplified as part of an amplicon with multiple other 8q-genes  and MYC (8q24.21). This amplicon contains 11 cancer relevant genes: BALC, ANGPT1, EIF3E, EBAG9, TRSP1, RAD21, EXT1, TNFRSF11B, NOV, HAS2, and RNF139 . The strongest correlations were observed for skin , pancreatic adenocarcinoma , ovarian cancer , liver hepatocellular carcinoma , and esophageal cancer  . Finally, we also observed that mRNA expression of multiple genes in the amplicon was also significantly increased  . All othncreased .RRM2B, MYC, and several other 8q-amplicon genes was increased, we next tested if the products of these genes within the 8q-amplicon interact with RRM2B. Since the expression of RRM2B amplifications are associated with specific tumor mutational signatures. For this analysis, we used the recent PanCancer Atlas data, reporting mutational spectra for 9493 individual tumors, including 926 BC, 384 OC, and 461 HNSCC and 524 LUAD . Additionally, RRM2B amplification cases tend to have worse DFS than MYC only cases (p = 0.0519).Based on the distinct tumor signatures observed in  cancers , we nextobserved , which iobserved . Howeverobserved . MYC ampRRM2B amplifications separately in each of the three major subtypes of breast cancer . We observed that patients with ER + PR + HER2 + BC bearing RRM2B amplifications had a significant decrease in OS  had significantly worse OS than ER- PR- HER2- patients (n = 26) (p = 0.0426). The previous studies that observed worse OS in BC, based on RRM2B amplification, did not consider subtype differences, and may have only included the major BC subtype (ER + PR + HER2 +). Additionally, p-values for all comparisons (log-rank test and Gehan-Breslow-Wilcoxon test) in all the above BCs are available in To further explore the conflicting findings in BC, we analyzed 2 + n = 8, ER + PRRRM2B, which is a major downstream target of p53, is highly amplified across multiple tumor types. Frequent RRM2B amplifications are a contrast to TP53 loss of function mutations or deletions that are typically found in tumors. We found that RRM2B, which is present on chromosome 8, is usually co-amplified with MYC oncogene . We also found, in multiple cancers, that tumors with co-amplified RRM2B and MYC exhibit significant amplification of the 8q22.3–8q24 region of chromosome 8. Intriguingly, we observed that several genes within the 8q-amplicon also interact with the RRM2B-functional network. Pathway enrichment analysis suggests the importance of 8q-region genes in response to oxygen levels, DNA replication, cell cycle, and DNA damage response.This study demonstrates that CDKN1A, GADD45, MDM2  a larger sample size of RRM2B-only amplified tumors and more balanced number of samples across each category, (b) analysis of outcomes in other cancers, and (c) other independent prognostic factors of overall survival. A previous study in breast cancer found frequent MYC co-amplifications with multiple genes in the 8q chromosomal region , and oxidative DNA damage. A previous study in mice found that overexpression of RRM genes combined with defective mismatch repair can lead to mutagenesis, and carcinogenesis (RRM2B overexpression on the levels of reactive oxygen species is not known. The observation of mutation signatures associated with increased reactive oxygen species suggests that RRM2B overexpression may impede the oxidative stress or oxygen sensing pathway. Finally, these observed mutation signatures suggest therapies that target defective DNA repair, such as PARP inhibitor therapy, may lead to clinical benefit in patients with RRM2B-amplified tumors (Mutation signature analysis revealed that ogenesis . While aogenesis , the impd tumors .in silico analysis of RRM2B alterations in cancer and their potential significance. Further studies in cellular models are warranted to delineate the role of RRM2B, and other 8q-chromosome genes in cancer cell maintenance, therapeutic targeting and clinical outcomes.Alterations in genes involved in DNA replication and/or repair, and DNA damage response pathways are known to increase genetic instability, and lead to cancer . RRM2B iRRM2B alterations in multiple tumor types, majorly reflected as RRM2B amplifications in conjunction with MYC, and other genes on chromosome 8. These cases exhibit a distinct 8q-amplification pattern as well as survival outcome differences and mutation signature differences, depending on cancer type and subtypes. Future genome-wide studies of other cancer datasets are warranted to confirm the results of the present study. Finally, studies in cellular models are required to delineate the role of RRM2B, and other 8q-chromosome genes in cancer cell maintenance, therapeutic targeting and clinical outcomes.Overall, this study provides an in-depth analysis of www.cbioportal.org.Publicly available datasets were analyzed in this study. This data can be found here: WI and ED: methodology, formal analysis, investigation, roles, writing—original draft, data curation, visualization, and validation. SS: formal analysis, investigation, roles, writing—original draft, and data curation. TV: software, formal analysis, and data curation, and visualization. GR: writing—review and editing, methodology, supervision, and funding acquisition. SA: conceptualization, methodology, writing—review and editing, supervision, project administration, resources, and funding acquisition. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Inflammation is the body’s defense reaction in response to stimulations and is the basis of various physiological and pathological processes. However, chronic inflammation is undesirable and closely related to the occurrence and development of diseases. The ocean gives birth to unique and diverse bioactive substances, which have gained special attention and been a focus for anti-inflammatory drug development. So far, numerous promising bioactive substances have been obtained from various marine organisms such as marine bacteria and fungi, sponges, algae, and coral. This review covers 71 bioactive substances described during 2015–2020, including the structures (65 of which), species sources, evaluation models and anti-inflammatory activities of these substances. This review aims to provide some reference for the research progress of marine-organism-derived anti-inflammatory metabolites and give more research impetus for their conversion to novel anti-inflammatory drugs. Inflammation is a kind of defensive response when the body is affected by various inflammatory factors or local injuries, and it is an important protective mechanism of the biological body . InflammThe ocean is where life is born and nurtured. It covers about 70% of the earth’s surface and 90% of the biosphere. The ocean has special physical and chemical conditions, including high salinity and weak alkalinity; the depths encompass an environment that is dark, cold, subject to high pressures, and presents many other complex characteristics . To bettMultiple signaling pathways, including nuclear factor-κB (NF-κB), Janus kinases/signal transducers and activators of transcription (JAK-STAT) and mitogen-activated protein kinase (MAPK) are involved in the regulation of inflammatory response, and play an essential role in a series of physiological and pathological processes in the body.2 (PGE2)) -1-phenazinecarboxylic acid (cid (21) , were is30 μg/mL .22)  and B (24) , four unusual 2, 3-dihydro-1H-indene isomers and a novel isoprenylisobenzofuran A (29), were separated from Diaporthe sp. SYSU-HQ3. Compounds 25–29   was isol 5.39 μM . Two natd B (24) , were isectively . Diaporids 25–29  exhibiteo 9.0 μM .1/15 of all marine animal species. Sponge has become one of the most abundant marine organisms in the discovery of marine active substances and represents an excellent resource for marine drug exploitation. To date, approximately 84 anti-inflammatory compounds have been isolated from marine sponges [Sponges, as the most primitive multicellular animals, have been living in the ocean for around 600 million years. To date, more than 10,000 types of sponges have been discovered, accounting for about  sponges . The ant30a)  [tertbutyl ether analogue of SA  (30a) (EC50 = 12 μM) with little cytotoxicity at up to 200 μM [1-tBuSA (30c)  and the expression of iNOS (EC50 = 20 μM) in LPS-stimulated RAW264.7 cells [Stylissatin A (SA) (30a) , a proli= 87 μM) . FurtherSA, 30b)  were appo 200 μM . A recenSA (30c)  could in.7 cells .31) and B (32)  in LPS-stimulated THP-1 cells; however, neither showed significant effects on the production of monocyte chemotactic protein 1 and TNFα [Dysidea septosa. Septosone A (33)  . An in vivo study showed that topical use of compound 34 significantly decreased mouse ear edema [35) (Dysidea arenaria exhibited inhibitory activities against COX-2 expression and PGE2 production in LPS-stimulated RAW264.7 macrophages [Dactylospongins A (d B (32)  are new and TNFα . Three me A (33)  indicatef 6.8 μM . The 9,1 μM [34)  extractear edema . The dysema [35)  isolatedrophages .36) and C (37)    are new ammation . A new pion [38) , was obtge cells .Marine algae are the oldest existing lower cryptogamous plants, with a wide variety of species . At present, four groups of seaweeds have been extensively exploited, including blue algae, red algae, brown algae, and green algae. Marine algae are known to be a rich source of bioactive metabolites and interesting pharmacological substances. The search for bioactive metabolites from seaweed has been very active . The antSolieria filiformis reduced neutrophil migration in a peritonitis model and decreased paw edema induced by carrageenan, dextran, and serotonin with no signs of systemic damage in mice [Caulerpa cupressoides var. lycopodium was investigated and showed that it decreased the carrageenan-induced rat paw edema and neutrophilic infiltration at 0.1, 1 or 10 mg/kg, and inhibited the expression of IL-1β, IL-6, TNFα and COX-2 at 1 mg/kg [Marine lectins are glycoproteins or peptides that bind to specific mono or oligosaccharides, which can promote cell recognition and adhesion, and some of them also showed strong anti-inflammatory activity. A lectin from the red marine alga  in mice . The ant 1 mg/kg .2 but did not change the IL-12/25 production, indicating that locally applied fucoidan might suppress airway inflammation in viral infections [Fucus vesiculosus L.  showed remarkable anti-inflammatory activity through the inhibition of COX-1/2, hyaluronidase and MAPK p38 [Turbinaria ornate (sulfate content 27%) displayed anti-inflammatory potential that could suppress NO production (IC50 = 30.83 ± 1.02 μg·mL−1) and dose-dependently reduce iNOS, COX-2, and pro-inflammatory cytokines including PGE2 levels in LPS-induced RAW264.7 macrophages and inhibit the production of NO and ROS in LPS-induced zebrafish embryo [Turbinaria ornata, a brown alga of the Sargassaceae family, is rich in bioactive molecules with various biological activities. The sulfated polysaccharide isolated from T. ornate could significantly reduce the paw volume and arthritic score in complete Freund’s adjuvant induced arthritis in rats. Interestingly, the sulfated polysaccharide could alleviate inflammation and bone damage at a low dose (5 mg/kg), indicating its potential in the management of chronic inflammatory diseases [Polysaccharides are the main components of marine algae, which have attracted much attention because of their various health benefits . Certainfections . The higMAPK p38 . The purh embryo . Turbinadiseases .39)  in LPS-induced macrophage cells [40)  [41) and M (42) and cystone C (43)  ether  , isolatege cells . Furtherge cells . A merodlls [40) , from thnd COX2) . Three ne C (43) , were alrophages .44)  in LPS-stimulated RAW 264.7 cells and can relieve inflammatory stress and suppress the expression of COX-2 and iNOS in LPS-stimulated zebrafish embryos [45) (Dictyopterismembranacea and displayed strong inhibition of NO production in LPS-induced RAW264.7 macrophages [46)   derived  embryos . A new dyos [45)  was obtarophages . The antges [46)  isolatedand IκBα .Coral is a large group of invertebrates belonging to the phylum Cnidaria, which is a low primitive organism with a wide distribution and with a wide variety of species (about 7000 known at time of writing). Coral is a marine biological resource that can be used extensively, in particular, soft corals and Gorgonians have been ranked highly with regard to the discovery of bioactive metabolites with potential pharmaceutical applications . In rece47)–along with a known analogue   [49)  -induced human neutrophils [51) (Sarcophyton stellatum showed anti-inflammatory activity by reducing the expressions of COX-2 and iNOS in LPS-stimulated mouse RAW 264.7 macrophage cells [52) and B (53))  , were obf 10 μM) . A naturμM) [49) , isolaterophages . Furtherltration . A new cion [50) , was obttrophils . A diterils [51) , isolatege cells . Two cem B (53))  were isoectively .54), uprolide O (55) and uprolide P (56)  and B (58)  , were isrophages . Two newd B (58) , were isated DCs .59) and B (60))   ) , were is80 μg/mL . A stero/mL [61)  obtainedtrophils . A new pils [62)  isolatedtrophils .63) (Cladiella hirsute and displayed strong anti-inflammatory activity in fMLF/CB-induced human neutrophils [64) and B (65)  , was obttrophils . Glaucumd B (65)  from Sarrophages .Inflammation, especially chronic inflammation, is a crucial contributor to the development of various human diseases. Regulation of inflammation to maintain its normal level is a key step in the treatment of related diseases. Although existing steroidal and non-steroidal anti-inflammatory drugs contribute a great deal, long-term use often causes adverse effects, including gastrointestinal discomfort, liver and kidney dysfunction, damage to the cardiovascular system, endocrine system, and so on. Marine organisms offer hope for the development of safe and effective new anti-inflammatory drugs. This review was conducted to provide reference for the research progress and give more impetus for the conversion of marine-organism-derived natural products to anti-inflammatory drugs. The Web of Science (WOS), PubMed, ScienceDirect, SpringerLink, and ACS databases were used for the preparation of the review, and some keywords such as “anti-inflammatory activity, natural product, marine bacteria and fungi, sponges, algae, and coral, etc.” were used for the search of relevant information. Finally, 71 bioactive substances described during 2015–2020 were presented, including the structures (65 of which), species sources, evaluation models and anti-inflammatory activities. Furthermore, some limitations could be obtained in this review: although a wide coverage was expected to be achieved, it’s extremely difficult to cover all the relevant literatures in view of the huge richness and diversity of marine organisms and their natural products; furthermore, a certain degree of randomness indeed exists for the presentation of the relevant literatures in the research field.Research into anti-inflammatory drugs derived from marine organisms started relatively late, but it has developed rapidly. As reviewed here, many anti-inflammatory substances have been obtained from a wide variety of marine organisms, including marine bacteria and fungi, sponges, algae, and corals. Preliminary studies have been conducted on their anti-inflammatory activities and mechanisms. Of course, we also need to be aware that the development and application of marine drugs still face many challenges. First, the extreme environment in which marine organisms live is difficult to simulate in the laboratory, which makes it extremely difficult to cultivate marine organisms and obtain large quantities of their active ingredients. Furthermore, the clinical effect and market application of some marine active substances remain uncertain due to their own limitations. For instance, although bioactive peptides have many well-known advantages, their clinical effects are often unable to match experimental results from the laboratory due to their complex structure, low concentration of active components, and closed N-terminal. Finally, a thorough safety assessment is crucial, as small differences in the amount used may lead to a shift in the role of the active products between poison and therapeutic.In future, we should try to investigate two aspects of this research: (1) take the isolated anti-inflammatory active substances from marine organisms as lead compounds, conduct functional modifications thereof, and study their structure-function relationship, so as to screen anti-inflammatory drugs with better efficacy; (2) strengthen resource integration, establish a comprehensive and efficient technological platform integrating detection, fermentation culture, separation and purification, functional modification, and effect evaluation, thus improving the efficiency of the development and application of new anti-inflammatory drugs. The ocean is a vast treasure trove, and there are still many bioactive compounds that have not been exploited. More extensive and in-depth studies should be conducted to find other, potentially valuable, marine drugs.


BCL11B, CCNK, YY1, DYNC1H1, and PACS2, were associated with the clinical phenotypes in our cases. Although the parents had normal chromosomes, two affected cases carrying terminal duplication of 14q32 can be explained by gonadal mosaicism. Further studies are needed to establish the association between cerebrovascular events and terminal duplication of chromosome 14q32, including investigation into the cytogenetics of patients with precise clinical descriptions.The terminal 14q32 duplication has been reported often in association with other cytogenetic abnormalities, and individuals with this specific duplication showed varying degrees of developmental delay/intellectual disability (DD/ID) and growth retardation (GR), and distinct facial dysmorphisms. Herein, based on the limited cases of terminal duplication of 14q32 known to date, we present new affected siblings presenting with DD/ID, GR, and facial dysmorphism, as well as cerebral infarction caused by recurrent de novo der(14)t leading to terminal duplication of 14q32. We used coverage analysis generated via duo exome sequencing, performed chromosomal microarray (CMA) as a confirmatory test, and compared our findings with those reported previously. Coverage analysis generated via duo exome sequencing revealed a 17.2 Mb heterozygous duplication at chromosome 14q32.11-q32.33 with a Z ratio ranging between 0.5 and 1 in the proband and her elder brother. As a complementary method, CMA established a terminal duplication described as the arr[hg19]14q32.11q32.33x3 in the proband and her elder brother; however, the parents and other siblings showed normal karyotyping and no abnormal gain or loss of CMA results. Five candidate genes,  Chromosome 14 is an acrocentric chromosome. Its short arm consists of only of the satellite that is extremely gene-poor and involved in various chromosomal rearrangement, including translocation. The finished sequence of human chromosome 14 comprises 87,410,661 base pairs, representing 100% of its euchromatic portion, in a single continuous segment covering the entire long arm with no gaps. The segment contains between 800 and 1300 genes and constitutes nearly 3.5 percent of the total cellular DNA . ParticuTo date, the various segments of abnormal rearrangements in 14q have been reported ,47,48,49https://gatk.broadinstitute.org/hc/en-us; accessed on 22 August 2020). Interpretation of sequence variants was reviewed manually by medical laboratory geneticists based on the Joint Consensus Recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology standards and guidelines [https://www.ncbi.nlm.nih.gov/clinvar/; accessed on 2 April 2021) or Online Mendelian Inheritance in Man  databases. All candidate variants were confirmed visually using an Integrative Genomics Viewer followed by Sanger sequencing with ABI3500XL Genetic Analyzer . In addition, copy number variations (CNVs) are a source of human genetic alteration and have been reported previously as a genetic cause of unexplained DD/ID, GR, facial dysmorphism, and/or CI [To identify the possible genetic causes of CI, growth retardation, and/or ID in patients, the exomic DNA of two affected siblings were amplified using Agilent’s SureSelect XT Human All Exon v5 . Based on suspected neurodevelopmental diseases, paired-end sequencing was performed to identify the genetic alterations using the Illumina NextSeq 550 System  at the Green Cross Genome . Base calling, quality check, read alignment, variant calling, and variant annotation for functional interpretation of results were carried out according to GATK Best Practices workflow for germline short variant discovery , according to the manufacturers’ protocol. All the samples were matched with Human Genomic DNA reference . Data were acquired using the Agilent Feature Extraction software 12.0.2.2 and Agilent CytoGenomics 4.0 and inspected visually using the Agilent Genomic Workbench Software 7.0.4.0 and Agilent CytoGenomics 4.0. Genomic locations were mapped using the human genomic reference sequence GRCh37/hg19. CNVs were classified using the ADM-2 algorithm with a minimum size filter cut-off of 200 Kb for copy number gains or losses in the region, and a minimal absolute average log2 ratio of 0.25 as the cut-off, copy number gain (0.25), or loss (−0.25) for heterozygous regions.a and b in c in d in The 14-year-old female proband II-4 in A presenta–d in The proband’s elder brother II-3 in A visitedFRMD4A (NM_018027.4: c.2296G>A/p.Ala766Thr) located on chr10:13,699,293 related to corpus callosum, agenesis, with facial anomalies, cerebellar ataxia (OMIM ID: #616819), and TBX1 (NM_080647.1: c.1480C>G/p.Pro494Ala) located on chr22:19,754,382 related to conotruncal anomaly face syndrome (OMIM ID: #217095). On the other hand, coverage analysis generated via duo exome sequencing revealed a 17.2 Mb heterozygous duplication at chromosome 14q32.11-q32.33 with a Z ratio ranging between 0.5 and 1 in the proband and her elder brother  located on chr14:101,004,893 and AHNAK2  located on chr14:105,410,932.On-target yields of 3,293,954,491 and 3,127,800,131 reads were generated from the proband and her elder brother based on the quality of all sequences. The mean read depths (×) were 65 and 62, and the percentage of bases above 30× was 81% and 85%, respectively. As a result, heterozygous variants of the two candidate genes associated with DD/ID, GR, or facial dysmorphism were identified via duo exome sequencing in both the proband and her elder brother:  brother A. As a c brother B. To invhttps://decipher.sanger.ac.uk/; accessed on 2 April 2021), and approximately 20 cases of pure 14q duplication are reported in the literature [Chromosome 14, one of five acrocentric chromosomes in the human genome, contains a heterochromatic short arm carrying ribosomal RNA genes essentially, and a euchromatic long arm in which most of the protein-coding genes are located. Phenotypic heterogeneity has been observed in children with partial duplication of various chromosomal segments. Whenever a larger or more distal segment of the long arm was involved, clinical features tended to be more severe. However, it is not clear whether substantial phenotypic variations between patients were due in part to individual differences, environmental factors, and the size of the trisomic segment, or the associated partial trisomy ,56. Duplterature ,47,48,49terature ,47,48,49On the other hand, duplication of 14q occurs as a result of various chromosomal structural aberrations, such as translocation, tandem duplication, inversion, or insertion ,47,48,49FRMD4A and TBX1 variants on chromosomes 10 and 22, respectively, identified in both affected siblings were excluded as a cause of clinical manifestations because these genes act in an autosomal recessive manner. Second, sequence mutations in some genes located on 14q have been reported in disease syndromes  [BEGAIN and AHNAK2, located on this region have yet to be linked to clinical manifestations in both affected cases, and these two genes and their associated clinical symptoms were not described in OMIM or elsewhere. However, the distal region of chromosome 14q32 duplicated in our cases involves nearly 120 genes according to Ensembl Genome Browser, and duplicated segments between 14q32and 14q33 were frequently reported by DECIPHER’s genome database and ClinVar. As a result, several protein-coding genes associated with DD identified by DECIPHER’s genome browser were expected to be related to DD/ID, GR, and facial dysmorphism  [The clinical features of the present case were compared with those described in the literature to identify a minimal overlapping region characterized from a molecular genetics perspective, including disease-associated genes. First, heterozygous aurosis) , but theil 2021) . Five ouBCL11B gene located on chr14:99,169,286-99,272,196 (OMIM #618092) can induce DD/ID with speech delay, dysmorphic facies, and T-cell abnormalities [CCNK gene located on chr14:99,481,408-99,512,439 (OMIM #618147) can lead to DD/ID with hypertelorism and distinctive facies [YY1 gene located on chr14:100,239,143-100,282,787 (OMIM #617557) can cause Gabriele-de Vries syndrome, characterized by mild-to-profound DD/ID in all affected individuals and a broad spectrum of morphological and functional abnormalities [DYNC1H1 gene located on chr14:101,964,572-102,056,442 (OMIM #614563) can lead to autosomal dominant mental retardation-13 with cerebral cortical malformations or microcephaly [PACS2 gene located on chr14:105,300,717-105,398,146 (OMIM #618067) cause developmental and epileptic encephalopathy-66 with facial dysmorphism and cerebellar dysgenesis, characterized by the onset of various types of seizures in the first days or weeks of life [TECPR2 gene is 0 and 14 other genes are awaiting review   .MEG3 and MEG8) or paternal inherited (DLK1 and RTL1) [On the other hand, imprinted regions were identified at 14q32 in the duplicated segment. The imprinted genes may be maternal (nd RTL1) . Consistnd RTL1) . FurtherIn the present study, the proband with DD/ID, GR, and facial dysmorphism showed CI, and her elder brother carried a history of suspected perinatal CI, in contrast to previous reports. Although the parents had normal chromosomes, two affected cases carrying terminal duplication of 14q32 can be explained by gonadal mosaicism. The proportion of abnormal germ cells is determined by the mutations early in germ cell differentiation. Further studies are needed to establish the association between cerebrovascular events and terminal duplication of chromosome 14q32, including investigation into the cytogenetics of patients with precise clinical descriptions.


TETs and related genes , the cell-type dependency and genomic distribution of 5-hmC, and the functional role of 5-hmC in the epigenetic regulation of transcription. We showcase examples of studies on three major smoking-associated cancers, including lung, bladder, and colorectal cancers, to summarize the current state of knowledge, outstanding questions, and future direction in the field.5-hydroxymethylcytosine (5-hmC) was first detected in mammalian DNA five decades ago. However, it did not take center stage in the field of epigenetics until 2009, when ten-eleven translocation 1 (TET1) was found to oxidize 5-methylcytosine to 5-hmC, thus offering a long-awaited mechanism for active DNA demethylation. Since then, a remarkable body of research has implicated DNA hydroxymethylation in pluripotency, differentiation, neural system development, aging, and pathogenesis of numerous diseases, especially cancer. Here, we focus on DNA hydroxymethylation in smoking-associated carcinogenesis to highlight the diagnostic, therapeutic, and prognostic potentials of this epigenetic mark. We describe the significance of 5-hmC in DNA demethylation, the importance of substrates and cofactors in TET-mediated DNA hydroxymethylation, the regulation of  An estimated 1.8 million new cancer cases were diagnosed in the United States in 2020 . A stronWhile many cancers are caused by smoking, not all smokers will develop cancer . A wide 5-hydroxymethylcytosine (5-hmC) was first detected in mammalian DNA in 1972 . ResearcIn mammals, DNA demethylation occurs through two distinct pathways: (I) ‘active’ demethylation and (II) ‘passive’ demethylation ,24. ActiAs mentioned above, maintenance of DNA methylation is accomplished by DNMTs during DNA replication through methylation of hemimethylated CpG dinucleotides ,51. FailThe TET family of enzymes, TET1, TET2, and TET3, in mammalian cells are α-ketoglutarate (α-KG)-dependent dioxygenases ,32,54. T2+ as a cofactor to convert 5-mC to 5-hmC, 5-fC, and 5 caC and generate CO2 and succinate [3+ to Fe2+ [2+ [Similar to other α-KG-dependent dioxygenases, TET enzymes require α-KG  and molecular oxygen as substrates and Feuccinate ,61,62. M to Fe2+ . Changes to Fe2+ . Moreove to Fe2+ . Vitamin to Fe2+ ,66,67. AIDH1 and IDH2 mutations are heterozygous, and the mutant isoforms generate 2-hydroxyglutarate (2-HG) instead of α-KG [IDH1 or IDH2 promotes 5-hmC formation in cells [IDH2, as observed in certain types of cancer, is associated with decreased levels of 5-hmC [The tricarboxylic acid cycle (otherwise known as the ‘Krebs cycle’), enzyme cytosolic isocitrate dehydrogenase (IDH1), and its mitochondrial homolog (IDH2) are responsible for generating α-KG, making them an integral part of the pathway involved in TET-mediated 5-hmC production ,61. IDH1 of α-KG . The acc of α-KG ,69,70,71in cells . Conversof 5-hmC . Moreoveof 5-hmC , resultiof 5-hmC ,74. Impoof 5-hmC ,76. Collof 5-hmC TETs [TDG [TETs and TDG are long genes and encode large proteins , which can be targeted post-transcriptionally by miRNAs, particularly in cancer, where the dysregulation of diverse classes of miRNA is very common [TET-mediated oxidation reactions can be modulated by post-transcriptional and post-translational regulation of TETs . FollowiTETs ,85,86,87ETs [TDG ,83,84,88y common ,21,88. Ay common , acetylay common , phosphoy common , GlcNAcyy common ,93,94, ay common . Proteiny common  and proty common ,97.TET enzymes have a lower affinity for 5-hmC when compared to 5-mC . ConsistWhereas global 5-mC content is relatively consistent across normal adult tissues , 5-hmC content is moderately low  and varies greatly across tissues (between 0.03 and 0.7%) ,101,102.TET1 and TET2 are highly expressed in ESCs, which is consistent with the abundance of 5-hmC in these cells [Accumulating data show a prominent role for 5-hmC in the epigenetic regulation of gene expression ,109,110.se cells .Differences in cell type seem to be a main determinant of the complex relationship between 5-hmC and gene expression ,38. For 5-hmC may be involved in regulating gene expression through effects on diverse regulatory elements and processes ,116,121.TET1 forms a complex with polycomb repressive complex 2 (PRC2) at H3K27me3 positive regions in the ‘bivalent’ state , and TETTET2 gene are frequent events in these types of cancer [TET2 mutations are found in 20% of acute myeloid leukemias, 20% of myeloproliferative neoplasms, 45% of chronic myelomonocytic leukemia, and 15% of T-cell lymphomas [TET1 mutations are found at lower frequencies, TET3 mutations are infrequent and have been shown to cause embryonic lethality in mice, suggesting that these mutations may not be tolerated in vivo [The role of TET enzymes in human cancer has been most extensively studied in hematopoietic malignancies ,23,38. Mf cancer ,135,136.ymphomas ,23,38. Tymphomas ,139,140. in vivo . We refe in vivo ,145,146.IDH1 and IDH2 occur frequently in human cancers [IDH mutations may then promote malignant transformation [Since TET enzymes are α-KG-dependent, they rely on IDH enzymes, which catalyze the oxidative decarboxylation of isocitrate to α-KG ,54. Muta cancers ,148,149. cancers . Similar cancers ,76, can  cancers ,68. The  cancers . Changesormation ,151,152.TET or IDH genes have been found in some malignancies, they are not present in all tumor types [Although mutations in the or types ,148,149.or types ,23,24. Oor types ,90,153. or types ,154. Of or types ,62. Receor types ,90. It has also been demonstrated that hypoxia, a hallmark of cancer, can varyingly change 5-hmC levels in different cell types ,24. In rCigarette smoke contains several thousand chemicals of which many act as oxidants, pro-inflammatory agents, carcinogens, and tumor promoters . Owing tAn emerging area of interest is the role of DNA hydroxymethylation modulated by smoking-induced oxidative stress in the initiation and progression of cancer . A recenWhile additional data are becoming available ,171 and Lung cancer is the leading cause of cancer-related deaths worldwide ,3. In thSeveral studies have investigated the genome-wide patterns of 5-hmC in tumor specimens from lung cancer patients . Li et aWang et al.  have anaTwo independent studies have quantified 5-hmC in the plasma cell-free DNA (cfDNA) of lung cancer patients as compared to controls. The first study by Song et al.  evaluateThe second study on the quantification of 5-hmC in the plasma cfDNA of lung cancer patients by Zhang et al.  employedTET1 by the C/EBPα transcription factor. Of note, oncogenic EGFR is found in approximately 15% of lung adenocarcinomas and several other cancer types [TET1 knockdown confers resistance to EGFR inhibitors in lung cancer cells. Loss of TET1 expression or mislocalized cytoplasmic TET1 is detectable in a substantial percentage of patient-derived lung cancer samples (~44%), suggesting that TET1 is likely inhibited in lung cancer [TET1 function can lead to the demethylation-dependent inactivation of tumor suppressor genes in lung cancer as well as adversely affect patients’ response to therapeutic options.Furthermore, mechanistic studies have investigated the role of TET enzymes in lung tumorigenesis. Forloni et al.  have reper types ,184,185.er types . They alg cancer . AltogetIn the United States, urinary bladder cancer is the fourth most common cancer in men, with an estimated 81,400 new cases and 17,980 deaths in 2020 ,187,188.Peng et al.  investigMunari et al.  utilizedTET1 mRNA to increase its protein expression. Upregulated TET1 then directly demethylates the ubiquitin specific peptidase 28 (USP28) promoter, thereby enhancing USP28 transcription and expression. Binding of USP28 to CD44 standard (CD44s) protein leads to removal of the ubiquitin group from the ubiquitinated CD44s protein, resulting in the stabilization of CD44s protein to mediate the stem-like property of human bladder cancer cells. Furthermore, CD44s inhibits RhoGDIβ degradation, which in turn promotes human bladder cancer invasion and lung metastasis [The role of TET enzymes in bladder cancer progression and metastasis has been investigated in multiple studies. Zhu et al.  identifitastasis . TP53 mutations are found in nearly half of all transitional cell carcinomas of the bladder, most frequently in high-grade invasive tumors [TP53 tumor suppressor gene and promotes its expression in bladder cancer cells (T24), whereas XIST inhibits the expression of TP53 by binding to TET1. Knockdown of XIST significantly suppresses cell proliferation and migration and induces apoptosis in bladder cancer cells, whereas overexpression of XIST has the opposite effects. The TP53-mediated cell proliferation and migration and apoptosis in bladder cancer were suggested to be modulated by XIST-related inhibition of TET1 binding to TP53 promoter, resulting in hypermethylation and decreased expression of this tumor suppressor gene [Hu et al.  reportede tumors . The autsor gene . Colon and rectal  cancers are the third leading sites of new cancer and the second deadliest malignancy, with an estimated 1.9 million incidence cases and 0.9 million deaths worldwide in 2020 . In the n = 97; paired tumors and normal colonic tissues), as well as patients with predisposing conditions, including benign polyps/colon adenomas  and inflammatory bowel disease  [TET1 and TET2 mRNA in CRC and AD samples were significantly lower than those in normal colonic tissues. Reduced expressions of TET1 and TET2 proteins  in CRC than normal colonic tissues were also confirmed by IHC analysis. No changes in expression of TET3 at mRNA or protein level were observed in the analyzed samples [The roles of 5-hmC and TET enzymes in the initiation and progression of colorectal cancer have been investigated as a means to identify diagnostic and prognostic biomarkers for this disease . Dziaman n = 49) ,204,205. n = 49) , in the  n = 49) , which a n = 49) ,204,205, n = 49) ,167,168. n = 49) . This ma samples . VAV2 and GNA12 by TET-assisted bisulfite sequencing (TAB-seq) confirmed that the gain of 5-hmC is accompanied by demethylation at the binding sites of these oncogenes. As HNF4A binding sites have been associated with regions losing methylation in intestinal differentiation [TET1 knockdown alters the expression of genes coding for proteins targeted to the cell membrane and extracellular space, thus inhibiting barrier formation of colonocytes. Comparison of genomic regions covered by 5-hmC in differentiated T84 cells in vitro and primary human colonocytes (n = 2) revealed a similar pattern of distribution, whilst a direct correlation was found between gene-specific 5-hmC changes and alterations in gene expression in human colon cancer tissues [Chapman et al.  investigntiation , the obs tissues .n = 90). Global 5-hmC levels were markedly decreased (on average an 85% drop) in the gDNA of tumors as compared to adjacent healthy tissues, as determined by ultra-sensitive capillary electrophoresis–electrospray ionization–mass spectrometry. A more limited decrease in global 5-hmC levels was detected in the cfDNA of colorectal cancer patients as compared to healthy controls, consistent with the low proportions of tumor-derived DNA in the total cfDNA pool. In the cfDNA of cancer patients as compared to controls, 5-hmC was enriched within gene bodies and DNase I sensitive sites, whereas it was depleted at TSS, CpG islands, and TF binding sites relative to the flanking regions. This implies that 5-hmC accumulates at positions surrounding TFs at active transcription sites. This was confirmed in the same samples where 5-hmC was enriched in regions marked by permissive histone modifications, such as H3K27ac, H3K4me1, and H3K9me1, whereas it was underrepresented in regions marked by the repressive modification H3K9me3. The average 5-hmC profiles of cfDNA were distinct from those of tissue and white blood cell gDNA, which might be ascribed to the different cells of origin and/or varying extent of DNA degradation in cell-free circulation. Among gDNA 5-hmC profiles, variations attributable to tissue identity (colon tissues vs. white blood cells) were dominant over variations related to disease status . Presumably, when gDNA from tumor tissue is released into plasma and diluted with the large quantities of background cfDNA derived from various other tissues, the tumor signal detected at a given locus is determined by the order of locus-, tissue-, and disease-specific variations. A model-based classifier using differentially hydroxymethylated loci identified in cfDNA and gDNA was developed, which predicted disease status, with high sensitivity (80–88%) and specificity (83–100%) in independent subpopulations of patients and healthy controls. This classifier also performed better than the conventional biomarkers (including carcinoembryonic antigen (CEA), alpha-fetoprotein (AFP), carbohydrate antigens , cytokeratin 19, and neuron-specific enolase (NSE)) and epidemiological risk factors  when predicting colorectal cancer. RNA-seq analysis of two tumors from colorectal cancer patients and paired adjacent tissues showed a significant correlation between changes in gene expression levels and alterations in 5-hmC levels in gene bodies in tumor samples (p = 9.8 × 106). DAVID pathway analysis revealed that genes with altered 5-hmC levels in tumor gDNA or cancer cfDNA were enriched in cancer- and metastasis-related pathways [Li et al.  performepathways .TET1, TET2, and TET3 in primary cancerous and histopathologically unchanged colorectal tissues from a cohort of 113 patients. Quantitative RT-PCR analysis showed significantly reduced TET1, TET2, and TET3 transcript levels in cancerous tissues as compared to histopathologically unchanged tissues. Importantly, patients with high TET2 mRNA levels in histopathologically unchanged tissues had favorable overall survival and disease-free survival outcomes. This implies that TET2 mRNA levels may have prognostic potential for CRC patients’ relapse and survival. Furthermore, bisulfite sequencing confirmed promoter hypermethylation in TET1 in cancerous tissues as compared to histopathologically unchanged tissues in a small portion of patients (12/113 = 10.6%). No DNA methylation was detected in cancerous or histopathologically unchanged tissues at the TET2 and TET3 promoters [Rawluzko-Wieczorek et al.  investigromoters . TET1 mRNA and 5-hmC in eight pairs of primary colon cancers and adjacent healthy tissues. Strong reduction in both TET1 transcript and 5-hmC levels was detected in cancer tissues as compared to controls by RT-qPCR and immunodot blot assays, respectively. Downregulation of TET1 was independent of tumor stage and the histopathological grade. The authors confirmed this finding by analyzing a metadata set of colon, breast, lung, and rectum primary tumors of varying stages (stages I to IV). Analysis of the genes differentially expressed in this cohort of 887 adenocarcinomas revealed that TET1, but not TET2 or TET3, was strongly downregulated in tumors since stage I. This indicates that TET1 downregulation is an early event in colon tumorigenesis. Additional experiments showed that TET1 expression and 5-hmC levels were easily detectable in human colon tissues and in normal epithelial colon cells (CCD), whereas no detectable levels of TET1 transcript or 5-hmC were observed in five different colorectal cancer cell lines . Silencing of TET1 in the normal CCD cells using two different shRNAs resulted in increased cell proliferation, suggesting that TET1 has a role in the control of cell growth. Rescuing experiments showed that re-expression of TET1 in the TET-silenced cells induced a full recovery of 5-hmC level and reduced cell proliferation rate. To demonstrate the effects of TET1 re-expression on the growth of colon cancer cells, Caco-2 and SW48 human colorectal carcinoma cell lines stably expressing TET1 under the control of a doxycycline (DOX)-inducible promoter were generated. Of note, re-expression of TET1 did not alter TET2 or TET3 expression in these cells. Upon treatment of the cells with DOX, elevation of 5-hmC levels concomitant with a strong reduction in growth rates were observed in both cell lines. These effects were due to the enzymatic activity of TET1, as the catalytically dead mutant (hydroxylase-deficient mutant TET1-H1672Y/D1674A) was not able to interfere with cell growth in vitro. To investigate whether TET1 plays a role in tumor growth in vivo, nude mice were injected with Caco-2 and SW48 cell lines and subsequently treated with DOX to induce TET1 expression. The size and weight of xenografts expressing TET1 were markedly smaller than those in the control group, wherein TET1 was not induced. Growth of the tumor xenografts was blocked not only when TET1 was induced early after the tumor cells’ inoculation (with both cell lines) but also several days afterwards when the tumors were already established. Functional experiments showed that TET1 inhibits cancer cells’ growth by repressing the WNT signaling pathway via demethylation of the promoters of the WNT inhibitors, Dickkopf Homolog 3 (DKK3) and Dickkopf Homolog 4 (DKK4), as reflected by the increase in 5-hmC and decrease in 5-mC levels in promoters of the respective genes in DOX-treated Caco-2 and SW48 cells as compared to controls. Rescue experiments in colon cancer cell lines in which the DKK3 and DKK4 genes were silenced and TET1 was re-expressed showed that knockdown of both genes restored the cell growth inhibition by TET1 expression [TET1 reactivation, although challenging, may represent a novel therapeutic approach for colon cancer and other types of malignancy.Neri et al.  analyzedpression . CollectIn 2009, the remarkable rebirth of an old epigenetic mark, discovered 37 years earlier , untanglGlobal depletion of 5-hmC together with locus-specific gain/loss of hydroxymethylation and variation in TETs activity and function, which are highly cell-type dependent, are hallmarks of cancer, as observed in solid tumors from patients with smoking-associated malignancies and in in vitro or in vivo systems ,23,24. TAn area of interest is the use of non- or minimally invasive surrogate tissues, which would faithfully inform the occurrence of epigenetic changes in target organs of smoking-associated cancers. In addition to conventional surrogate tissues, such as peripheral blood, oral epithelial cells, and urine ,212,213,As we continue to investigate the role of DNA hydroxymethylation in the initiation and progression of smoking-associated cancers in patient populations, we should also expand our studies to healthy populations of smokers and nonsmokers. The latter studies, particularly with a prospective design, should facilitate identification and validation of diagnostic and prognostic biomarkers of smoking-associated cancers. With the same token, the growing population of electronic nicotine delivery system (ENDS) users, whose alternative tobacco products are known to contain toxic and carcinogenic compounds  ,222,223,