Macaca mulatta, was cloned and its immunological characteristics were preliminarily explored. The results showed that Macaca mulatta TRIM4 was in the same branch as human TRIM4. Overexpression of TRIM4 increased the mRNA levels of interferon (IFN)-alpha, IFN-beta, RIG-I, MAVS, IRF3, IRF7, OAS1, IFIT3, and CCL5, TRIM4 up-regulated the activities of IFN-beta, NF-κB, and ISRE reporter. In contrast, inhibiting TRIM4 expression by small interfering RNA (siRNA) down-regulated the IFN pathway. In summary, Macaca mulatta TRIM4 plays an essential role in the IFN pathway.Monkey diseases are becoming increasingly severe, and some may be transmitted to humans through direct and indirect contact. Innate immunity is the first line of defense against foreign microorganisms. It is of great significance to explore the immune characteristics of monkey and human diseases. TRIM4, an important immune molecule in  Innate immunity plays an important role in the resistance to foreign pathogen invasion. Foreign microorganisms are recognized by pattern recognition receptors (PRRs), including toll-like receptors (TLRs), NOD-like receptors (NLRs), RIG-I-like receptors (RLRs), and C-type lectin receptors (CLRs) . The bodTripartite motif protein (TRIM) is a vital signaling molecule that plays an essential role in many cellular processes, including cell proliferation, differentiation, carcinogenesis, innate immunity, and apoptosis . For exaTRIM4 gene was cloned from rhesus macaque (Macaca mulata), and the relationship between TRIM4 and IFN was preliminarily explored, providing a theoretical basis for treating diseases.Studies have shown that human TRIM4, an essential regulatory molecule of RIG-I, participates in the interferon (IFN) pathway . However2. There was no mycoplasma contamination in the MARC145 cells.MARC145 cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM)  together with 10% fetal bovine serum  at 37°C in a humidified atmosphere containing 5% COMacaca mulatta was provided by Guangzhou Xusheng Biotechnology Co. Ltd. The ethics committee of Foshan University has approved all animal experiments. Macaca mulatta liver samples were used for RNA extraction with TRIzol reagent . HiScript III 1st Strand cDNA Synthesis Kit (+gDNA wiper)  was used for reverse transcription. Finally, complementary DNA (cDNA) was used for subsequent molecular cloning experiments.Xho I and BamH I, PCR product was ligated to pCMV-3xFLAG-7.1. The primers of TRIM4 were shown in TRIM4 upstream and downstream primers were designed according to the CDS region of the TRIM4 sequence, the Xho I restriction site was incorporated in the upstream primer, the downstream primer contains a BamH I restriction site. The 2× phanta master mix  was used for the PCR amplification, and reaction conditions were 95°C for 5 min, followed by 40 cycles at 95°C for 30 s, 53°C for 30 s and 72°C for 120 s. The Mix & Go! E. coli Transformation Kit  was used for transformation. PurePlasmid Mini Kit  was used for plasmid extraction.TRIM4 cloning was performed using a standard procedure for molecular cloning. After double digestion with The phylogenetic tree was constructed using the neighbor-joining method with MEGA 5.0 .The plasmids, poly(I:C)  and poly(dA:dT)  were transfected using Lipofectamine 3000 Transfection Reagent .Macaca mulatta TRIM4 siRNA (si-TRIM4) and non-targeting control siRNA (si-NC) were transfected into MARC145 cells using RNAi-mate . The si-TRIM4 and si-NC were synthesized by GenePharma Co., Ltd. Knockdown efficiency was verified by western blotting.FLAG-tagged TRIM4 (FlAG-TRIM4) plasmid (300 ng) or si-TRIM4 (60 nM), reporter plasmid (200 ng), and RL-TK (20 ng)  were transfected into MARC145 cells using Lipofectamine 3000. After 48 h, reporter activity was measured by a luciferase reporter assay . The IFN-beta, nuclear factor-κB (NF-κB), and ISRE reporter plasmid are bought from Beyotime  and kept in the laboratory as previously described .IFN-alpha, IFN-beta, retinoic-acid–inducible gene I (RIG-I), mitochondria antiviral signaling protein (MAVS), interferon regulatory factor (IRF) 3, IRF7, 2′-5′ Oligoadenylate synthetases (OAS)1, interferon-induced protein with tetratricopeptide repeats (IFIT3), and CCL5 were determined by real-time PCR on a 7500 Fast Real-time PCR system . The PCR procedure and quantification were performed as described in a previous study  for 1 h. Then, the medium was changed to DMEM with 2% FBS, and samples were obtained at the indicated time points.MARC145 cells were infected with the PRRSV-2 BJ-4 strain  were used to analyze the data. Values of p < 0.05 were considered statistically significant.There are three biological repeats in all the experiments. Mean ± standard deviation is the method of displaying data, and paired student's Macaca mulata TRIM4 gene from healthy Macaca mulata liver tissue. TRIM4 gene is 1,425 bp in length and encodes 475 amino acids. Phylogenetic tree analysis showed that Macaca mulata TRIM4 is in the same branch as humans and pan troglodytes, and they share a close genetic relationship  control group was set up. First, TRIM4 was well expressed in cells  for 9 h, and the promoter activities were tested. The results showed that the activities of IFN-beta, NF-κB, and ISRE promoters decreased significantly after transfection with TRIM4 siRNA . The results showed that 0.01, 0.1, and 1 MOI PRRSV infection increased the Macaca mulata TRIM4 mRNA levels and protein levels (To investigate the effect of porcine reproductive and respiratory syndrome virus (PRRSV) infection on n levels . The chan levels . Furthern levels .TRIM4 in 9 h  increased the expression of 4 in 9 h . In addi4 in 9 h . The res4 in 9 h . In addi4 in 9 h .Viral pathogen-associated molecular patterns (PAMPs) are recognized by PRRs, inducing type I IFN and downstream proteins, which play a pivotal role in initiating the antiviral immune response. Type I IFN mediated by PRRs is regulated by post-translational modifications, especially phosphorylation and ubiquitination .Macaca mulata TRIM4 regulates immune response is still unknown.TRIM protein is expressed in cellular physiological processes, such as cell differentiation, apoptosis, tumor genesis, and innate immunity . An incrIFN-alpha, IFN-beta, RIG-I, MAVS, IRF3, IRF7, OAS1, IFIT3, and CCL5; and TRIM4 knockdown decreases the activity of IFN-beta, NF-κB, and ISRE promoters, and decreases transcription levels of IFN-alpha, IFN-beta, RIG-I, MAVS, IRF3, IRF7, OAS1, IFIT3, and CCL5. These results reveal a critical role of monkey TRIM4 in the immune response against viruses.Our study confirmed that TRIM4 is a positive regulator of the type I IFN induction pathway. Overexpression of TRIM4 activates IFN-beta, NF-κB, and ISRE promoters; TRIM4 increases transcription levels of Macaca mulata TRIM4 were reported first in this study. The above experiments further confirmed that Macaca mulata TRIM4 is closely genetically related to human TRIM4, and monkeys are a practical animal model to study human diseases. Sequence analysis showed that TRIM4 has the RING, B-Box, and coil-coil domain; these indicate that Macaca mulata TRIM4 may be functionally similar to human TRIM4.The immunological characteristics and nucleic acid information regarding Expression of TRIM4 increases after PRRSV infection in MARC145 cells, indicating that TRIM4 plays a role in the immune regulation of PRRSV. PRRSV activates the expression of TRIM4, prompting an immune response in the host and inhibiting virus replication. The results showed that TRIM4 overexpression inhibits PRRSV replication, indicating that TRIM4 played an antiviral role as a host antiviral factor.TRIM4 increased, which indicated that TRIM4 played a role in the immune regulation of poly(I:C). These results indicate that TRIM4 responses to RNA virus regulation, the poly(I:C) results coincide with PRRSV results.Poly(I:C) is a simulated RNA analog, and after poly(I:C) stimulation, the expression of TRIM4 increased, which indicated that TRIM4 functions in the immune regulation of poly(dA: dT). These results indicate that TRIM4 functions in the regulation of DNA viruses.Poly(dA: dT) is a simulated DNA analog, and after poly(dA: dT) stimulation, the expression of Macaca mulata TRIM4 may contribute to similar regulatory mechanisms and participate in the monkey IFN pathway. Macaca mulata TRIM4 may play an antiviral role, and these mechanisms will be studied in the future.Studies on TRIM4 have shown that TRIM4 interacts with transient receptor potential melastatin 8 and regulates its channel function through K423-mediated ubiquitination . TRIM4 cMacaca mulata TRIM4 is a crucial regulatory molecule in the IFN pathway and functions a vital role in revealing the monkey immune response.In summary, Macaca mulata TRIM4 positively regulates IFN-beta in the IFN pathway.The original contributions presented in the study are included in the article/The animal study was reviewed and approved by Foshan University.MZ, HL, and HZ performed the experiments and wrote the manuscript. HS analyzed the data. LH and RW conceived and designed the experiments. All authors read and approved the final manuscript.This work received funds from the National Natural Science Foundation of China . The funder had no role in the study design and collection, analysis, and interpretation of the results.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.


Cancer immunotherapy using monoclonal antibodies targeting immune checkpoint proteins, such as PD-L1 or PD-1 , have demonstrated improved overall survival in several human cancers. For example, a recent phase III clinical trial (JAVELIN Bladder 100) demonstrated that bladder cancer patients treated with Avelumab (anti-PD-L1 antibody) have significant improvement in both overall survival and progression-free survival as compared to a control group [Recently, studies have suggested that cancer growth reprograms the epigenome, which may lead to escape from immunosurveillance. For example, epigenetic modifications, namely DNA methylation and histon modification H3K27me3, have been shown to suppress the transcription of TH1-type trafficking chemokines CXCL9 and CXCL10 in human cancer . EpigeneEpigenomes, Lee et al. summarized several recent findings that epi-drugs were found to have immunomodulatory properties [In the Special Issue of the journal operties . For exaoperties ,7, whileoperties ,9. Inhiboperties . Furtheroperties .It must be noted that more than 50% of the human genome is made of transposable elements (TEs), which are highly repetitive DNA sequences . The TEsIn this regard, several clinical trials involving combination treatment of epi-drugs and ICB are underway in several human cancers, including breast cancer ,13. Epig


O-methylation of specialized metabolites in plants is a unique decoration that provides structural and functional diversity of the metabolites with changes in chemical properties and intracellular localizations. The O-methylation of flavonoids, which is a class of plant specialized metabolites, promotes their antimicrobial activities and liposolubility. Flavonoid O-methyltransferases (FOMTs), which are responsible for the O-methylation process of the flavonoid aglycone, generally accept a broad range of substrates across flavones, flavonols and lignin precursors, with different substrate preferences. Therefore, the characterization of FOMTs with the physiology roles of methoxylated flavonoids is useful for crop improvement and metabolic engineering. In this review, we summarized the chemodiversity and physiology roles of methoxylated flavonoids, which were already reported, and we performed a cross-species comparison to illustrate an overview of diversification and conserved catalytic sites of the flavonoid O-methyltransferases. The  O-methylation reactions. The O-methylation of flavonoid aglycones results in reduced molecular activity of a hydroxyl moiety and increased lipophilicity, and it modifies their intracellular compartmentation. Furthermore, O-methylation provides a branchpoint in the biosynthesis of diverse metabolic pathways, such as lignin and flavonoid phytoalexin biosyntheses, which promote antimicrobial activity [O-methylation stabilizes the B ring, increasing its water solubility and enhancing its redshift effect [Pueraria lobata, yellow lupin and alfalfa roots, respectively [O-methylation or glucuronidation, which reduces their ability to donate hydrogen atoms [Flavonoids are a large group of phenolic compounds; this group encompasses flavonol, flavanone, flavone, isoflavone, flavanol and anthocyanin subgroups, which play important roles in the physiological response of plants to various biotic and abiotic stresses. The core structure of flavonoids, their “aglycone”, is composed of A, B and C rings, whilst a series of modification reactions, such as hydroxylation, glycosylation, prenylation and methylation, can enhance multiple physiological functions corresponding to both their structural diversity and tissue specificities. The hydroxylation reaction binds to the backbone in different numbers and positions, being the basis of subsequent activity ,2. In tht effect . In termt effect ,5, and 3ectively ,7,8. Howen atoms . In the en atoms , as wellen atoms . Furtheren atoms . The bioen atoms ,13.S-adenosyl-methionine (SAM)-dependent methyltransferases are enzymes utilizing SAM as a universal methyl donor, which operates in both primary and secondary metabolism [O-, N- or C- methyltransferases . Plant OMTs act as hydroxyl group acceptors of small molecules, such as flavonoids, alkaloids, phytoalexins, lignin precursors, simple phenols and phytohormones [O-methyltransferases (FOMTs) of plants belong to class I and class II subgroups according to their molecular weight and cation dependency. The class I caffeoyl-CoA OMT (CCoAOMT) type group is of a lower subunit molecular weight (26–30 KDa) and displays cation-dependent activity. The enzymes of this subclass mostly participate in lignin, phytohormone and scent metabolism. Such class I CCoAOMT genes are, therefore, thought to have evolved under the pressure of terrestrial colonization [P. deltoides × P. nigra), phylogenetic analysis combined with comparative genomics of OMTs indicated that OMTs have evolved by gene duplication; COMT genes, COMT-like genes and several FOMTs are thought to have evolved different functions or tissue specificity via tandem gene duplication followed by subsequent diversification [Brachypodium distachyon, three duplicated genes encoding COMT are proposed as species-specific functional genes, since these COMTs do not locate in collinear genomic regions of rice and sorghum. Functional characterization verifies that they have lost the original catalytic activity toward caffeic acid [Vanilla planifolia, two COMT-like genes that evolved from a COMT gene have a novel function in catalyzing methyl gallate and flavone luteolin production [tabolism . Based ohormones . Flavononization . The enznization . For thinization . In hybrfication . In Braceic acid . In Vanioduction . Althougoduction ,21,22,23With the exception of a minority of OMTs, which exhibit strict enzymatic specificity for a single substrate, plant OMTs generally accept a broad range of substrates across flavonoids, lignin precursors and alkaloids, albeit with different substrate preferences ,24. SuchO-methylation can occur at any position; however, 7- and 4′-methoxylation are the most highly represented since their corresponding hydroxyl moieties are attached during the biosynthesis of the flavonoid aglycone  , 1598 flavonoids were found to be methoxylated flavonoid derivatives    found inIn our survey and data mining, Fabaceae, Asteraceae, Lamiaceae and Rutaceae were the top four families in terms of the diversity of methoxylated flavonoids. As for the distribution of each group of methoxylated flavonoids, Asteraceae (25%), Fabaceae (15%), Lamiaceae (4%), Rutaceae (3%), Brassicaceae (2%) and Ericaceae (2%) occupied almost 50% of the variety of methoxylated flavonols . In the Medicago, Trifolium, the Cicer genus and Glycine max , in 272 Fabaceae species were found in KNApSAcK. By combining the taxonomy relationship and metabolite information, the isoflavones with a mono-methoxyl group were found to be the most widely distributed, especially in some common studied species, such as cine max . The 6-mO-methylation of anthocyanin increases its water solubility, strengthens its color properties and shifts the color to being more red based on the methylation level [O-methylation produces the major pigment subgroups peonidin, petunidin and malvidin, which are responsible for a purple appearance. Malvidin- and petunidin-type anthocyanins have been found to accumulate in colored grape berries, cyclamen flowers, petunia flowers, purple tomato seedlings, Nemophila menziesii blue flowers and torenia blue petals [The flavonoid pathway is thought to be ancient and to have evolved during adaption from an aquatic to terrestrial habitat, thus being supposed to be involved in various physiology processes and complex stress defenses ranging from coloration to anti-pathogen activity and symbiosis . Flower on level . Moreovee petals ,36,37,38e petals ,40. Givee petals .O-methylation on the enhancement of antimicrobial activity has consistently been reported [OsAldOMT1 [Lotus corniculatus, Medicago sativa, Pisum sativum and Trifolium pratense [MsIOMT overexpression line increased the resistance of alfalfa to the leaf pathogen Phoma medicaginis [Aspergillus oryzae and Rhizopus oligosporus in soybean seedlings as opposed to daidzin, which accumulated in the negative control [O-methxylation of naringenin, rapidly responded both to UV irradiation and phytopathogen infection [Based on the biological functions of flavonoids, the effect of reported . To promsAldOMT1 . The metpratense ,44,45,46icaginis . Glyciti control . Furthernfection ,48. M. sativa and Vicia sativa are indicated as members of potential inducer factors [M. truncatula supports the important role of the nodule infection zone expression gene chalcone 2′-OMT and its corresponding product 4,4’ -dihydroxy-2’-methoxychalcone in the symbiosis process [Dittrichia viscosa contain apigenin, 6-methoxykaempferol, rhamnetin (7-methoxyquercetin), isorhamnetin (3′-methoxyquercetin) and dihydroxyquercetin; 7-methoxykaempferol and 6-methoxykaempferol reduce the root length and root biomass of lettuce seedlings, respectively [O-methylation on symbiosis and pathogen interaction essentially comes from indirect evidence and needs to be subjected to systematic research. In addition, the effects of environmental conditions, including temperature, nutrition and water sufficiency, on flavonoid O-methylation remains to be explored.Methoxylated flavonoids have additionally been proposed to affect the interaction with symbiotic bacteria and plants. From the summary of ‘infection flavonoids’, which induce the Nod factor in the nodulation process in legume plants, 7- or 4′-methoxygenistins, glycitin, apigenin in soybean root and methoxychalcone in  factors . Further process . Moreove process ,52. Acco process . Currentectively . The staectively . These rO-methyltransferases, which methylate different positions or substrates, are constantly being isolated [Glycyrrhiza echinata, Hordeum vulgare, Mentha × piperita, Oryza sativa, M. truncatula, Populus deltoids and Ocimum basilicum are involved in the catalysis of a variety of substrates, including isoflavones, luteolin, apigenin, quercetin and naringenin [O. sativa and H. vulgare, OsNOMT and HvF7OMT were found, following pathogen inoculation, to methylate naringenin and apigenin, respectively. In M. truncatula, although MtIOMT1, MtIOMT2, MtIOMT3 and MtIOMT7 all modify the 7-O- position, they have different substrate preferences with glycitein, daidzein, 6,7,4′-trihydroxyisoflavone and naringenin, respectively [Corresponding to methoxylated flavonoid diversity, flavonoid-isolated . For flaringenin ,59,60,61StF3OMT isolated from Serratula tinctorial displayed a preference for quercetin, followed by kaempferol and myricetin, with no requirement for Mg2+ [SlMOMT3 and ShMOMT3 were characterized to catalyze the methylation of the aglycone, as well as the 7/3′/4′ methoxylated form of quercetin, kaempferol and myricetin, which participate in a series of methylation reactions leading to the highly methoxylated myricetin present in trichomes [CrOMT1 isolated from Catharanthus roseus was found to display a border preference for phenylpropanoids, such as 5-hydroxyferulate, in addition to the 3-O- position methylation of flavonols [Lupinus luteus roots [CdFOMT5, which catalyzes 3,3′,5,7 methylation of flavones in citrus fruit peels [Given the requirement for 3-hydroxyl group existence, substrates of F3OMT are limited to flavonol aglycones. The for Mg2+ . In wildlavonols . With reit peels .PFOMT was the first isolated F6OMT, which could methylate diverse flavonols and caffeoyl-CoA derivatives but only those with a vicinal dihydroxyl moiety. Phylogenetic analysis of protein sequences suggested that the PFOMT gene diverged from CCoAOMT genes. The subunit molecular weight, which ranges from 26 to 30 KDa (estimated as 26.6 KDa), and its Mg2+-dependent reaction indicated PFOMT to be a class I OMT, providing the clue that F6OMT might differ from flavonol/flavone OMTs decorating other positions, which usually belong to class II OMTs [Plagiochasma appendiculatum and Glycine max, the PFOMT-like proteins PaF6OMT and GmIOMT1 were identified as F6OMT catalyzing the production of scutellarein, baicalein and 6-OH daidzein, respectively. The characteristics of the small subunit size being estimated as 27.4 KDa and 26.75 KDa and their activity being cation dependent are consistent with belonging to PFOMT [O. basilicum, two ObF6OMTs actually shared high similarity with F4′OMTs; their protein mass and non-cation reaction indicated them to be class II OMTs [MpOMT2, was isolated to catalyze the 8-hydroxy-7-methoxyflavone in M. × piperita [O. basilicum also isolated the homologous gene of MpOMT2 designated as ObF8OMT-1, both of which were cation-independent OMTs. Interestingly, the other class I OMT gene was designated as ObPFOMT, which displayed cation-dependent activity and required a vicinal hydroxyl group. It was additionally demonstrated to display an 8 methylation activity to flavone in O. basilicum [F6OMT and F8OMT are rarely isolated because of the rare existence of 6/8 methoxylated flavonoids requiring additional hydroxylation on these positions. The ice plant  II OMTs . Severalto PFOMT ,64. Acco II OMTs . As for piperita . Subsequasilicum .MtIOMT5, HI4′OMT, PIOMT9 and SOMT2, corresponding to the production of 4′-methoxylated isoflavones, have been isolated from the legumes M. truncatula, G. echinate, P. lobate and G. max [MpOMT4 from M. × piperita, flavone apigenin 4′OMT (PaF4′OMT) from P. appendiculatum and flavanone homoeriodictyol-4′OMT (CrOMT6) from C. roseus were also characterized [ShMOMT2 was isolated from wild tomato trichome methylates from the 4′-O- and 7-O- positions of flavonol to produce trichome-specific methoxylated myricetins  [In the B ring of flavonoids, 4′-methoxylation often corresponds to anti-pathogen activity. In Fabaceae, 4′-methoxylated isoflavones (formononetin and biochain A) are methoxylated isoflavones, and they are induced by pathogen infection. Several F4′OMTs, such as d G. max ,59,66,67d G. max ,68. Besicterized ,69,70. Mricetin) . O-methylation and specificities for flavone luteolin and flavonol quercetin in vitro. However, AtCCoAOMT7 had promiscuous positional selectivity for flavanones, eriodictyol and dihydroquercetin, which led to a mixture of a specific ratio of 3′-O-methylether and 4′-O-methylether [PFOMT from ice plant also had both 6-OMT activity and 3′-OMT activity with quercetagetin as substrate [AtOMT1 is involved in multiple biosynthetic pathways, including flavonol, lignin, melatonin and NAD biosyntheses. AtOMT1 displays enzymatic activity with 3′-O-methylation of quercetin [O-methylation activity with caffeic aldehyde, 5-O-hydroxy-ferulic acid, 5-O-hydroxyconiferaldehyde and caffeoyl alcohol [AtOMT1 also displayed weak N-methylation activity with nicotinate [N-acetylserotonin methylation activity [The 3′/5′OMTs are widely found, with the most well known being the production of isorhamnetin by quercetin 3′-OMT. The CCoAOMT-like protein AtCCoAOMT7 of Arabidopsis displayed substrate specificity with a preference for 3′-hylether . The PFOubstrate . Similarubstrate ,74,75. Bubstrate ,77,78,79uercetin  and also alcohol ,82. In acotinate  and in vactivity . VvAOMT could only catalyze glycosylated anthocyanins and flavonols and not aglycones. The types of glycosides attached to the substrate also affected the relative specific activity of VvAOMT [PsAOMT mediated color spot formation by methylating cyanidin-3-O-glucoside to the darker peonidin-3-O-glucoside [SlAnOMT could produce petunidin glucoside utilizing delphinidin 3-O-glucoside. Silencing SlAnOMT in fruits and hypocotyls resulted in a reduction in the content of petunidin and malvidin [GmOMT5 was characterized as a pigment isogene methylating cyanidin glucoside to peonidin glucoside in black seed coated compared to brown seed coated soybean seeds [NmAMT3, NmAMT6 and MT2 lotus from Nemophila menziesii and Petunia x hybrida, also display slight 3′,5′ catalytic activity as well as 3′-OMT function [In the case of anthocyanin, F3′OMTs and F3′,5′OMTs have been much characterized in studies of flower color engineering. By contrast to the other FOMTs, which prefer aglycone substrates, anthocyanidin OMTs prefer glycosylated substrates. In grape, F3′,5′OMT f VvAOMT . In peonlucoside . In purpmalvidin . In resean seeds . Besidesfunction .O-position. The isoliquiritigenin 2′-OMT from alfalfa has been postulated to play a role in the nodulation process due to it producing the Nod gene inducer 4,4’-dihydroxy-2’-methoxychalcone in the rhizobia infection area of root hairs [Humulus lupulus, which is used as an ingredient for beer brewing, a chalcone 6′-OMT  OMT1 was characterized to produce the flavor compound xanthohumol from desmethylxanthohumol in trichomes [Given that chalcones are missing a C ring in their chemical structure, chalcone OMTs are a unique class methylated at the 2′-ot hairs ,86. Furtrichomes . CdOMT5 and CrOMT2 from C. depressa and C. reticulata were characterized to methylate at the 3,3′,5,7 and 3′,5′/7 positions with a wide range of flavones or flavonols, respectively [TaOMT1 and TaOMT2 were found to sequentially methylate tricetin via 3′-O-monomethyl ether (selgin) to 3′,5′-dimethyl ether (tricin) to 3′,4′,5′-trimethyl ether order [In addition to the FOMTs mentioned above (4.1 to 4.6), some FOMTs show methylation activity for multiple sites, especially in plant species that produce a high level of methoxylated flavonoids, such as citrus and wheat. The peel of citrus fruits contains high contents of methoxylated flavones, with up to seven methoxylation sites  . CdOMT5 ectively ,88. In wer order ,90. MtIOMT1-7 from M. truncatula; flavonol OMTs ShMOMT1-4 from S. habrochaites; flavone OMTs MpOMT1A, 1B to MpOMT4 from M. × piperita; and flavone OMTs ObFOMT1-6, ObF8OMT-1 from O. basilicum [M. truncatula elicited leaves, over-expressing MsI7OMT did not produce isoformononetin (7-methoxydaidzein) but rather accumulated formononetin (4′-methoxydaidzein) [O-methyltransferase, could also methylate the 7-O-position of kaempferol and quercetin aglycone in vitro; however, no 7-methoxylated flavonoids were detected in vivo, indicating that the only in vivo substrate of this enzyme was 10-hydroxycamptothecin [A set of FOMTs with different regioselectivity and substrate preferences have been characterized in same species, such as isoflavone OMTs asilicum ,58,59,79aidzein) . In camptothecin .GmI6OMT from soybean [The FOMTs belonging to class I and class II OMTs harbor conserved catalytic sites and substrate binding regions but are separately discussed according to their differences in length, conserved amino acid sites and cation interaction . In 1998 soybean . ObaCVOMT1, ObaEOMT1 and TaCOMT-3D [Of the class II OMTs, the chalcone OMT and isoflavone OMT from alfalfa were the first two reported crystal structures of plant OMTs, revealing their substrate specificity . The straCOMT-3D ,96.para- to meta-O-methylation of flavanones and dihydroflavonols in vitro [O-methyltransferase POMT7 protein allowed the methylation of both 3, 7-hydroxyl groups of quercetin instead of the 3-hydroxyl group alone [M. trunculata alter the substrate preference from a higher tricetin and 5HFA (5-hydroxyferulic acid) affinity to a higher 5HFA and tricetin affinity, respectively [M. truncatula characterized a set of FOMTs sharing high similarity; however, having a different substrate preference and regioselectivity can also provide clues for essential residue identification [Along with the increasing number of elucidated crystal structures of FOMTs, homology modeling optimized by known FOMTs with docked substrates provided a prediction of the possible residues that affect activity and selectivity. The Gly46 in AtCCoAOMT7, which is equivalent to Tyr51 in known PFOMTs, was indicated via docking studies to be a part of the substrate binding pocket close to the catalytic site. The mutation of Gly46 to Tyr led to a reverse in the in vitro  (Figure in vitro . Moreoveup alone . The Valectively . Moreovefication ,59. Besification . O-methyltransferase in wheat compared the homodimer and dissociated monomers with the dissociated monomer and concluded that the monomers retain their catalytic capacity [Thalictrum tuberosum showed selectivity to a variety of new substrates from catechols to hydroxycinnamates and alkaloids compared to its corresponding homodimers [Papaver somniferum [The protein crystal structures of PaMTH1, MsChOMT, MsIOMT and HI4′OMT suggest that the homodimer formation generated by the N-terminal swapping of FOMT protein forms the functional homodimers in solution or the cell ,102,103.capacity . Neverthmodimers . A recenmniferum . AccordiO-methylation of specialized metabolism in the plant kingdom. Flavonoids are phytochemicals involved in pathogen defense and UV light protection, and they enable plants to interact with their environment. Evolutionary analysis of flavonoid biosynthesis suggests that this pathway originated very early in plant colonization of land. However, the lignin biosynthesis pathway, which is also derived from phenylpropanoid metabolism, as well as flavonoid biosynthesis, is considered to be an essential factor of land colonization of plants. Given that many FOMTs have multiple preferences toward both flavonoid and lignin biosyntheses metabolites, the diversification and convergence of substrate selectivity and physiological functions involving multiple pathways are a topic for future research. Due to the multiple substrate selectivities caused by protein dimerization and the difference in activity performance in planta and in vitro, a comprehensive elucidation covering all substrate specificities and their interaction is very complex. Therefore, a comprehensive approach combining profiling data of endogenous methoxylated/non-methoxylated compounds and a gene expression analysis considering their tissue specificity and stress deducibility of FOMTs is required in future. In this review, we summarize our current knowledge of the chemical diversity and physiological roles of methoxylated flavonoids. Additionally, we provide a cross-species comparison of methoxylated flavonoids and FOMT genes. Such plant-species-wide approaches will give an overview of the diversification of the


Among its many effects, most prominent is its action to accelerate cell proliferation, thereby modulating wound healing. It also mediates affects in the gastrointestinal system, in the testes, and in secretory organs, including the pancreas, salivary, and prostate glands. On the cellular level, Zn2+ is involved in protein folding, DNA, and RNA synthesis, and in the function of numerous enzymes. In the mammary gland, Zn2+ accumulation in maternal milk is essential for supporting infant growth during the neonatal period. Importantly, Zn2+ signaling also has direct roles in controlling mammary gland development or, alternatively, involution. During breast cancer progression, accumulation or redistribution of Zn2+ occurs in the mammary gland, with aberrant Zn2+ signaling observed in the malignant cells. Here, we review the current understanding of the role of in Zn2+ the mammary gland, and the proteins controlling cellular Zn2+ homeostasis and signaling, including Zn2+ transporters and the Gq-coupled Zn2+ sensing receptor, ZnR/GPR39. Significant advances in our understanding of Zn2+ signaling in the normal mammary gland as well as in the context of breast cancer provides new avenues for identification of specific targets for breast cancer therapy.Zinc (Zn Zinc is a vital trace element present in all body tissues and organs. There are differences in recommendation of expert groups regarding the daily allowance of dietary zinc, with suggested intake of 10–20 mg/day, depending on age and gender , and low2+) act as structural components of many enzymes and transcription factors [2+ in proliferation, and, indeed, Zn2+ deficiency is associated with attenuation of cell growth [2+-depleted cells, while its supplementation enhanced genome stability even following radiation treatment [2+ has another important role: acting as a signaling molecule. This conclusion is supported by the large number of proteins involved in compartmentalization of the ion, allowing transient local changes in Zn2+ concentrations, as required for signaling [2+ levels and its interaction with cellular kinases or receptors induce what is termed Zn2+ signaling. An example of this is the interaction of Zn2+ with the growth hormone receptor, and its modulation of the insulin-like growth factor-I (IGF-1) pathway [2+ as a first messenger, regulating the ZnR/GPR39 G-protein coupled receptor, or as a second messenger, activating major pathways associated with proliferation [Zinc ions  or by Zn2+ transporters [2+ transport across cellular membranes, as Zrt- and Irt-related proteins (ZIP) mediate influx into the cytoplasm and Zn2+ transporters (ZnT) move Zn2+ out of the cytoplasm [2+ homeostasis is tightly regulated by these proteins under normal circumstances. Intriguingly, Zn2+ levels are dysregulated in the microenvironment surrounding cancer tissue [2+ transporters related to the presence of cytokines and growth factors in the tumor’s microenvironment [Under physiological conditions, cellular Znar range . Yet, “fservoirs ,35,36. Rsporters ,37,38. Tytoplasm . Zn2+ hor tissue . This fiironment .2+ is emerging as an important player in mammary gland function under both physiological and neoplastic conditions. We will discuss molecular mechanisms mediating the activities of Zn2+ in this tissue.Zn2+ to act as a signaling molecule, both its intracellular and extracellular levels must transiently change. In addition, there must be protein targets that detect these changes and subsequently trigger downstream effects. On a fast scale, intracellular Zn2+ transients involve a Zn2+ release from intracellular stores, e.g., endoplasmic reticulum or Zn2+-binding metallothioneins, in response to stimuli [2+ influx that upregulates mitogen-activated kinase (MAPK). This, in turn, activates the release of sequestered Zn2+, thereby raising the intracellular Zn2+ concentration [2+ levels are produced by its release from stores following epidermal growth factor (EGF) stimulation, which induces casein kinase 2 phosphorylation of a Zn2+ transporter, ZIP7 [2+ transporter activity [2+ signaling events, occurring several hours after cell stimulation, and are dependent upon changes in expression of Zn2+ transporters that modulate the transfer of Zn2+ from one compartment to another [2+ rises place this ion in the category of classical second messenger, directly regulating major signaling pathways. Among those pathways activated by Zn2+ are EGFR [2+ levels can also modulate signaling via inhibition of phosphatases activity [For Zn stimuli ,41,42. Fntration . Similarer, ZIP7 . In addiactivity ,43. Late another ,37. Bothare EGFR , the IGFare EGFR , and MAPare EGFR . Changesactivity .2+ levels can also transiently increase, as many cell types secrete Zn2+ from intracellular vesicles to the extracellular region. Such release is documented in neurons, pituitary cells, prostate epithelial cells, mast cells, granulocytes, Paneth cells in the intestines, pancreatic cells, and mammary gland epithelia [2+ can then interact with cell surface proteins that have Zn2+-binding modulatory sites, including ion channels, neurotransmitter transporters, and G protein-coupled receptors [2+ induce transactivation of EGFR in airway epithelial cells [2+, acting as a first messenger in mouse embryonic stem cells (ES), induces proliferation through upregulation of PI3K, MAPK, and mTOR signaling pathways [2+ and triggers metabotropic calcium (Ca2+) release [2+ is the ligand of the orphan receptor GPR39 [Extracellular Znpithelia ,50,51,52eceptors ,54,55. Fal cells , as wellal cells ,58. Thuspathways . Signifi release . Later, or GPR39 , and we or GPR39 ,62.2+ to mammary gland development is manifested by the severe consequences of Zn2+ deficiency. Under these conditions, extreme defects in morphology and function, including oxidative stress and inflammation, are described in the non-lactating [2+, as described above [2+ is suggested to regulate proliferation and differentiation of the epithelial cells via its interaction with kinases downstream to the prolactin receptor. Mammary gland involution, following lactation, is also highly dependent on Zn2+ that acts as a regulator of apoptosis via mitochondrial or lysosomal pathways [2+ transporter, ZnT2, is essential for accumulation of Zn2+ in lysosomes and assembly of the vacuolar ATPase for initiation of mammary gland involution [The importance of Znactating ,64 as weactating . During actating . Interesed above . During ed above . At thispathways ,70. The 2+ to support its rapid growth and development. Mammary cells are tasked with providing this important nutrient, which they do at a rate of about 1 mg Zn2+ per day during lactation [2+ deficiency to the infant, leading to, among other things, growth retardation and skin lesions [2+ deficiency, diminished cognitive development is observed [2+ is found in secretory vesicles in the mammary gland and its loss is associated with a condition termed neonatal Zn2+ deficiency [2+ from the circulation into the mammary cells is therefore crucial, and its concentration in milk is maintained over a wide range of maternal dietary Zn2+ intake [2+ in the ingested milk, several Zn2+ transporters are present in the mammary tissue , which degrade extracellular matrix components and are essential for remodeling of the tissue during pregnancy and involution [2+ transporters, ZnT5 and ZnT6, are expressed on the Golgi apparatus and play a prominent role in activation of Zn2+-binding enzymes, such as the tissue non-specific alkaline phosphatase (TNAP) [2+-deficient maternal milk, as well as low TNAP activity in mothers of severely symptomatic neonates [A developing offspring requires relatively large amounts of Znactation ,14,71. F lesions ,72. Evenobserved ,74. Indeficiency . During + intake ,76,77,78y tissue , providiactation . A pointthe milk . Additio infants ,82,83. Z infants ,81,84. Dvesicles . Studiesr, MTF-1 ,86. Follysosomes ,87,88. Svolution . The Zn2e (TNAP) ,90. Inteneonates .2+, several Zn2+ importers from the ZIP family have been identified in the mammary epithelium  attenuated migration of these cells . Correspand ZIP6 . Heterodand ZIP6 , which cand ZIP6 . Knockdoand ZIP6 . An elegand ZIP6 . The Zn2 mitosis .2+-sensing Gq-protein-coupled receptor (ZnR/GPR39) acts as a link between extracellular Zn2+ and intracellular pathways that regulate cell proliferation and survival [2+ and mediates metabotropic Ca2+ signaling via the IP3 pathway in colonocytes, keratinocytes, and neurons [2+ interacts with ZnR/GPR39 and initiates cell signaling events that trigger release of intracellular Ca2+ [The selective Znsurvival ,146. Thesurvival . The fir neurons ,148,149.lar Ca2+ ,61,150.2+ signaling, ZnR/GPR39 activates MAPK via extracellular signal-regulated kinase (ERK1/2), and the phosphoinositide-3 (PI3)-kinase pathway [2+-dependent manner, accelerated wound closure in HaCaT keratinocytes [2+ levels in the prostate are high [2+ concentration is decreased [2+ binding to it will activate Ca2+ signaling, leading to proliferation. Further evidence for the role of ZnR/GPR39 in cancer comes from a study of esophageal squamous cell carcinoma (ESCC) shows that overexpression of GPR39 is associated with lymph node metastasis [2+, increases cell proliferation [Downstream to the Ca pathway , both cl pathway . Indeed,inocytes . A similinocytes . Howeverinocytes . Interesare high , and expecreased ,97, ZnR/tastasis . In contferation . Furtherferation .+ transport by the Na+/H+ exchanger (NHE) [2+ on NHE activity and attenuation of the recovery from acidification [+/H+ exchange, it also acts as a scaffold protein by recruiting cytoskeletal linker proteins, PI3K and AKT, thereby triggering cell growth [2+ and ZnR/GPR39 in cell proliferation and migration.More evidence of a role for ZnR/GPR39 activation in cancer progression comes from work showing that it regulates several important ion transporters thought to play a role in cell survival and proliferation. In neurons and colonocytes, activation of ZnR/GPR39 enhanced Her (NHE) ,155. ZnRer (NHE) . Furtherfication . In kerafication . Upregulfication . It has fication . Moreovefication . In addil growth ,161. It +/Cl− -cotransport (KCC) isoform 2 [− gradient and, thereby, GABAA currents in neurons [− absorption and decreased water loss in a diarrhea model. Other KCC family members are essential for regulating epithelial cell volume, a function linked to morphological changes underlying formation of plasma membrane protrusions involved in metastasis as well as cell proliferation [+/Cl- cotransporter KCC3, which may locally affect cell volume resulting in formation of protrusions [In hippocampal CA3 neurons, ZnR/GPR39 increases surface expression and activation of Ksoform 2 ,163. KCC neurons . Similar neurons  and resuferation . Studiesferation . A recentrusions . In additrusions , which d2+, among them are the Zn2+ transporters discussed above. Thus, a modulator of ZnR/GPR39 that will not trigger other Zn2+-dependent pathways is of clear importance.Development of the normal breast is mostly regulated by estrogen acting through the ER , thereby2+ homeostasis suggest that they may provide novel targets for cancer therapies. In addition, our work suggests that ZnR/GPR39 can activate critical compensatory pathways following the loss of hormone regulation of mammary cell growth. Thus, ZnR/GPR39 represents a highly viable target for new molecular or immune approaches to hormone-resistant cancers and TNBC.Zinc is an essential ion, required for cell function and proliferation. Less appreciated is its role in promoting oncogenesis and in metastasis. The important role of this ion in itself and of the transporters that are involved in maintaining cellular Zn


There is currently a lack of pharmaceuticals targeting neurodegenerative disorders via the regulation of phase separation. The potential of melatonin in the modulation of biomolecular condensate in the attenuation of aberrant condensate aggregation in neurodegenerative disorders is discussed in this review.Biomolecular condensates are membraneless organelles (MLOs) that form dynamic, chemically distinct subcellular compartments organizing macromolecules such as proteins, RNA, and DNA in unicellular prokaryotic bacteria and complex eukaryotic cells. Separated from surrounding environments, MLOs in the nucleoplasm, cytoplasm, and mitochondria assemble by liquid–liquid phase separation (LLPS) into transient, non-static, liquid-like droplets that regulate essential molecular functions. LLPS is primarily controlled by post-translational modifications (PTMs) that fine-tune the balance between attractive and repulsive charge states and/or binding motifs of proteins. Aberrant phase separation due to dysregulated membrane lipid rafts and/or PTMs, as well as the absence of adequate hydrotropic small molecules such as ATP, or the presence of specific RNA proteins can cause pathological protein aggregation in neurodegenerative disorders. Melatonin may exert a dominant influence over phase separation in biomolecular condensates by optimizing membrane and MLO interdependent reactions through stabilizing lipid raft domains, reducing line tension, and maintaining negative membrane curvature and fluidity. As a potent antioxidant, melatonin protects cardiolipin and other membrane lipids from peroxidation cascades, supporting protein trafficking, signaling, ion channel activities, and ATPase functionality during condensate coacervation or dissolution. Melatonin may even control condensate LLPS through PTM and balance mRNA- and RNA-binding protein composition by regulating N Present in all cells, biomolecular condensates are membraneless organelles (MLOs) containing proteins, ribonucleic acids (RNAs), and other nucleic acids . These mPhase separation at its core is a thermodynamic process driven by the reduction or a negative change in global free energy ,13. LLPSMutation, mis-regulated RNA processing, and the altered binding of RNA in MLOs that are enriched with RNA and RNA-binding proteins (RBPs)  often re16−22 and disrupted prefibril formations 1F0 ATP s [1F0, P —only F1Fform ATP ,130, wheform ATP , and V-tform ATP . Once bell lines ,133, endll lines , human ull lines , HepG2 [ll lines ,137 and ll lines , HaCaT kll lines , and evell lines .0 domain of both bacterial and eukaryotic ATP synthase is embedded within the plasma membrane, the hydrophilic, water-soluble catalytic F1 domain of eukaryotes is oriented towards extracellular space, whereas that of bacteria is directed inwards toward the cytoplasm [1F0 rotors during ATP synthesis [Although the Fytoplasm ,141. Botytoplasm ,145,146.ytoplasm ,148,149.ynthesis .1F0 ATP synthase on isolated rat hepatocytes plasma membrane was independently confirmed a few years later [2+-ATPases [2+ ATPase (PMCA) were found to be exclusively localized to cholesterol/sphingomyelin-rich lipid raft domains of caveolae in pig cerebellum synaptic plasma membranes [2+ ATPases (SERCA) were similarly identified in caveolae/lipid rafts in human uterine cells [For the first time, in 2004, ATP synthase alpha and beta were discovered to be expressed in lipid rafts isolated from rat HepG2 hepatocytes by immunofluorescence , and a frs later . In the rs later . P-type -ATPases  such as embranes , and sarne cells , rat hepne cells , and humne cells . Capablene cells , caveolane cells ,160,161.ne cells ,163,164,ne cells . In ordene cells ,167. Thene cells ,152 is r1F0 ATP synthases have been observed to translocate from mitochondria to lipid raft domains of various cell types, including plasma membranes of gonadotropes [The ATP synthases of mammalian mitochondria are usually arranged in rows of dimeric complexes of two identical monomers located at the highly curved apex of deep IMM invaginations known as cristae . Dimerizdotropes , and thedotropes .Biomolecular condensates adapt to changing endogenous or exogenous conditions  by conti1F0 ATP synthases in various human cancer cell lines translocated to cell surface lipid rafts or to lipid raft domains present in mitochondria [LLPS is further regulated by lipid raft membrane-anchored proteins that support the continuous restructuring and rearrangement of molecular components in condensates . Cell suchondria . The prechondria ,183 and chondria ,105 protchondria ,184. Faichondria  to functchondria , ATP synchondria  may contchondria  and cancchondria .Lipid bilayers in cell membranes are composed of hundreds of different lipid species with a propensity to segregate laterally into subcompartmentalized raft domains ,188. Fouo) and liquid-disordered (Ld) phases to induce elastic interactions by reducing line tension between Lo and Ld phases [o phase lipid raft domains [1 domains to cell surfaces while significantly doubling the degree of extracellular ATP production within 30 min of exposure [Line tension, or the energy required to create boundaries between lipid raft domains and the surrounding membranes, is one of the key drivers that can determine the size, form, and shape of physiological nanoscopic lipid compartments . The hydd phases ; in addi domains . Furtherexposure ,210.The ability of ATP synthase/ATPase to form dimerized rows on the IMM of mitochondria and other membrane surfaces may be highly dependent upon membrane lipid composition  and curvInability to neutralize excess reactive oxygen species (ROS) accumulated as products of normal cellular functions results in a state of imbalance often referred to as oxidative stress . Oxidati0 motor of the ATP synthase controls proton flux that powers the rotation of the F1 subunit protruding into the mitochondrial matrix, driving the synthesis of ATP [1F0 ATP synthase with higher affinity than all other mitochondrial phospholipids [0 section of ATP synthase [Cristae are dynamic, independent, bioenergetic IMM invaginations capable of remodeling in seconds to organize respiratory chain supercomplex assembly and ATP synthase for efficient ATP production ,222. Mits of ATP . CL is rs of ATP ,234. Thiholipids . ATP synsynthase , whereassynthase . Mitochosynthase ; therefosynthase , includisynthase , nonalcosynthase , thyroidsynthase , diabetesynthase ,244, cansynthase , as wellsynthase , Parkinssynthase ,248, amysynthase , Barth ssynthase ,251, andsynthase ,253. Thesynthase ,255. Altsynthase ,257.Cardiolipin (CL) is a mitochondria signature lipid distinctly attracted to membrane lipid domains with strong negative curvatures, such as the apex of IMM cristae ,228. CL α-syn demonstrates a high affinity for mitochondrial membranes, interacting in close proximity with mitochondrial OXPHOS proteins, including lipid raft-like domains at MAMs that are high in phospholipids ,283. NatPhysiological lipid rafts function optimally at nanoscopic sizes ,293,294.Saccharomyces cerevisiae under ROS attack [Melatonin is a potent antioxidant that has been shown to inhibit CL peroxidation in mitochondria, preventing mPTP opening and Cyt c release  by inhibS attack . The NLRS attack ,267,273.S attack ,305,306 ATP-dependent DEAD-box RNA helicases (DDXs) are ATPases that regulate RNA-containing phase-separated organelles in prokaryotes and eukaryotes ,308. DDXThe activation of the NLRP3 inflammasome is now associated with major neurodegenerative disorders such as AD, PD and ALS, where positive correlations have been found to exist between NLRP3 levels and abnormal protein aggregations such as Aβ and α-Syn, whereas the inhibition of the NLRP3 pathway attenuates pathological protein aggregations . MelatonNanoscopic transient lipid raft domains in biological membranes are formed by phase separation in response to external stimuli ,93,188. d phase to drive cholesterol into the ordered Lo phase [1–40 and Aβ1–42 peptides are known to interact strongly with negatively charged lipids by binding to anionic, negatively charged membranes [In 2005, melatonin was first observed to induce phase-separation in DPPC lipid bilayers ; recentlLo phase . These sembranes ,325,326.embranes . Althougembranes ,162, itsembranes ,330,331.25–35 peptides embedded in hydrocarbon cores of anionic lipid bilayers may further displace cholesterol molecules to increase oligomerization or fibrillation [In animal and in vitro studies, melatonin was able to prevent or ameliorate tau and Aβ pathology in AD ,334,335 illation , but theillation . Melatonillation . On the illation .Local variations in melatonin concentration also affected the re-ordering of lipids in membranes. At 0.5 mol% concentration, melatonin was documented to penetrate lipid bilayers to form fluid domains that enriched lipid membranes where melatonin molecules aligned parallel to phospholipid tails with the electron-dense regions slightly below hydrophilic headgroups; however, at 30 mol% concentration, melatonin molecules aligned parallel to the lipid bilayer, close to the headgroup regions where one melatonin molecule was associated with two lipid molecules to form an ordered, uniform, lateral membrane structure distributed evenly throughout the membrane model . VariatiMembrane fluidity reveals the degree of molecular disorder and motion within lipids in membrane bilayers . There ad phase [o–Ld phase separation over a range of temperatures and domain sizes, effectively preventing the formation of a non-raft Ld phase, possibly by reducing line tension or acting as a surfactant at Lo–Ld interfaces [It is believed that lipid rafts function optimally as nanodomains ,293, whed phase . Interesterfaces . ATP is terfaces ,31 capabterfaces . Whetherterfaces  to inducterfaces . NonetheDuring lipid peroxidation events, oxidized moieties were found to mainly reside close to the lipid headgroups forming hydrogen bonds with water. These oxidized lipids can perturb membrane bilayer structures and modify membrane properties, including decreasing the membrane fluidity ,354,355.Lipid rafts are phase-separated regions in lipid bilayers responsible for important biological functions including signal transduction ,93 as we+ as a result [Present in all eukaryotes , CYB5R3 a result ,371. Thea result , whereasa result ,373,374.a result ,376.Originally known as mitochondrial porin after its identification in yeast (1985)  and humaCYB5R3 enzymes form large redox centers in lipid rafts that enhance mitochondrial respiration rate and ATP production, albeit resulting in increased production of ROS ,373,374.VDAC is the gatekeeper which controls the export of ATP out of mitochondria into cytosol and the import of essential respiratory substrates such as ADP and Pi into mitochondria ,399; theThe mitochondrial electron transport chain is a major ROS-generating site where complex III and mitochondrial glycerol 3-phosphate dehydrogenase can produce large amounts of redox signaling molecules such as superoxide and hydrogen peroxide to the external side of the IMM as well as the matrix ,404. BisMelatonin is an ancient, potent antioxidant that protects lipid nanodomains from peroxidation caused by excess oxidative stress. The addition of micromolar concentrations of melatonin to rat heart mitochondria dramatically inhibited CL oxidation by tert-Butylhydroperoxide (t-BuOOH), a peroxidation promoting peroxide, reversing cytochrome c release, matrix swelling, and proton motive force (ΔΨ) collapse in treated cells . The melN-acetyl-5-methoxytryptamine) is a mitochondria-targeted molecule found in cells of all tested eukarya and bacteria [N-acetyltransferase (AANAT), the enzyme responsible for the rhythmic production and release of melatonin in bacteria, fungi, unicellular green algae, and chordates [Rhodospirillum rubrum and cyanobacteria, precursors to mitochondria and chloroplasts, respectively [Melatonin  in prokaryotic bacteria ,428, whehibitors .0CF1) control transmembrane electrochemical proton gradients for the production of ATP [The gene sequences of cyanobacteria ATP synthase subunits are extremely similar to those in chloroplasts . Embedden of ATP ,438,439.n of ATP , PG is tn of ATP . Both CLn of ATP ,445,446.•OH) through the sequestration of metal ions and deactivating •OH during Fenton-like reactions in Type II antioxidant reactions [Melatonin and its secondary, tertiary, and quaternary metabolites actively scavenge potent free radicals ,426,447 eactions . In addieactions .•OH employing density functional theory found that one molecule of melatonin effectively scavenged two •OH radicals to produce the stable footprint metabolite, cyclic 3-hydroxymelatonin (3-OHM) [•OOH) at rates 98.4 times faster than Trolox in aqueous solution [•OOH and other free radicals that penetrate deep into lipid molecules.A recent study that analyzed the mechanistic interactions between melatonin and  (3-OHM) , in perf (3-OHM) ,459,460.solution . Melatonsolution ; therefo•OH [•OOH, the two most prevalent ROS responsible for the chain oxidation of unsaturated phospholipids [All biological cell membranes comprise amphipathic lipid molecules with hydrophilic heads and hydrophobic tails that naturally form bilayers with headgroups oriented towards an aqueous environment and tails facing each other . The mel•OH  and •OOHholipids ,466 in tholipids ,468.•OOH and •OH and terminates with reactive aldehyde end products such as malondialdehyde (MDA) and 4-hydroxynonenal (HNE) [•OOH and •OH are derived from ubiquitous superoxide radicals (O2•−) generated from the one-electron reduction of oxygen (O2) that may be catalyzed by nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase) during respiratory bursts [−1·s−1 [2•− behaves more similarly to an unimpressive reductant (E°′(O2/O2•−) = −0.33 V) than an oxidant (E°′(O2•−/H2O2) = 0.93 V) [•OOH [•OOH or HO2•), also known as a perhydroxyl radical, is a chemically active, protonated form of superoxide radicals (O2•−) [2•− into hydrogen peroxide (H2O2) which then can further be transformed via Fenton’s/Haber–Weiss reactions [•OH, possibly the most reactive and mobile species of oxygen that interacts with almost all molecules in cells [•OOH exists primarily as the less reactive O2•−, where the ratio of protonated •OOH to anionic O2•− is ~130:1 (less than 1%), •OOH can be a potent initiator of lipid peroxidation [Lipid peroxidation, a physiological process in all aerobic cells , is a caal (HNE) ,472,473.y bursts  and/or ey bursts . Due to  [−1·s−1 , O2•− be 0.93 V) ,477,478 V) [•OOH ,479. Hyds (O2•−) , engagedeactions  into •OHin cells ,481. Evexidation ,466.•OOH to accumulate at lipid headgroup membrane–water interface at concentration enhancement of over one order of magnitude [•OH, •OOH, and H2O2 with polar headgroups of phospholipid bilayer revealed that all three species traveled deep into the water layer to reach phospholipid biomolecules, oxidizing hydrophilic headgroups before hydrophobic tails [•OOH staying adsorbed for the longest duration at headgroup regions [When reacting with phospholipids, the advantageous free energy profile of −8.5 kJ/mol free energy minimum relative to the aqueous phase allowed agnitude . Multi-lic tails , with •O regions . The hea regions , support regions . +) concentration (~700 to ~800) [+ concentration at membrane surfaces may cause accumulation of •OOH, the protonated form of O2•− [The strong negative curvature of cristae in the IMM is primarily sustained by the distinct molecular geometry of CL with its smaller, elongated, conical-shaped, double-phosphate dianonic headgroups that increase lateral pressure within the acyl chain regions and stabilize cylindrically curved, tubular cristae structures ,485,486.to ~800) , is the to ~800) . At the  of O2•− .•OOH remains adsorbed at polar headgroups longer than other ROS tested [S tested ; therefoS tested ,497,498 S tested  and/or uS tested . In the S tested ,501 becaS tested . The abiS tested .As early as 2002, melatonin was found to increase mitochondria OXPHOS activity and elevate the production of ATP . Recent Saccharomyces cerevisiae and human hepatocarcinoma cells challenged with hypoxic stress, the non-canonical RNA-binding proteins in glycolytic enzymes have been observed to promote phase separation [Interestingly, in paration  that facparation . Melatonparation ,504,505;paration . These rparation . Alteratparation . Increasparation ,517,518.Biomolecular condensates containing protein, RNA, and other nucleic acids  are formCells also employ post-translational modifications (PTMs) to induce non-equilibrium thermodynamic chemical reactions in order to tune the molecular interactions of key condensate components where external energy input drives reactions out of equilibrium to control the size and number of MLOs . PTMs, iCellular stress in eukaryotes activates defense mechanisms such as stress granules (SGs) that can promote either survival or apoptosis . IntegraUnder cellular stress conditions, phosphorylation can initiate the formation of SGs  and alsoMissense mutations associated with diseases are found mostly within IDRs . These mIntriguingly, ATP has recently been reported to exhibit a unique biphasic relationship with TDP-43 PLD. At a molar ratio of only 1:25 (PLD:ATP), TDP-43 PLD was induced to undergo LLPS to start forming liquid droplets in a dose-dependent manner where many droplets could be produced at a 1:100 molar ratio. Further increases in ATP, in contrast, led to a reduction in droplet formation. At 1:750, only a few droplets could be detected, and at 1:1000, all droplets were disassembled by ATP. Importantly, in the absence of ATP, TDP-43 PLD was unable to phase-separate into droplets . NeuronsMutations in fused in sarcoma (FUS) are associated with ALS pathology, and are believed to be a major cause behind familial ALS ,599. UndPhysiological FUS is a transcription factor  which haC. elegans, phosphorylation also promoted IDR granule disassembly, whereas dephosphorylation promoted granule assembly [Phosphorylation is one of the most important PTMs that can control the assembly/disassembly of MLOs  as well assembly . Under dassembly ,351,536,assembly ,538.The ATP-dependent DEAD-box helicase  DDX3X reAD is associated with the aggregation of Aβ as well as the intracellular deposition of neurofibrillary tangles (NFTs) of tau, a major neuronal protein with important physiological functions of stabilizing and promoting the assembly of microtubules (MTs) in the central nervous system (CNS) ,635. TheLLPS of tau has been demonstrated to promote amyloid aggregation . Tau canTau phosphorylation has been proposed as a neuroprotective mechanism  where ph1–42-induced memory impairment, synaptic disorder, and tau hyperphosphorylation-associated neurotoxicity in C57BL/6N mice [25–35 in pheochromocytoma (PC12) cells by activating melatonin-PTEN/Akt/GSK3β signaling [The use of melatonin in neurodegenerative disorders has been extensively studied and reviewed ,692,693./6N mice ; restori/6N mice ; and dec/6N mice . Melaton/6N mice . Even thignaling . The majignaling  adds an Even though melatonin is able to ameliorate tau-pathology-related symptoms such as oxidative stress, neuroinflammation, cognitive impairment, cell death, and tau hyperphosphorylation in vivo , the fac1–40 and Aβ1–42 to inhibit the formation of β-sheet and/or amyloid fibrils [1–40 and Aβ1–42 with only 100 μM of melatonin could not be replicated in control experiments using a potent free radical scavenger N-t-butyl-a-phenylnitrone (PBN), or a melatonin analog 5-hydroxy-N-acetyl-tryptamine (NAT) [An in vitro study on Neuro2A cells reported that melatonin at 10 μM concentration reduced intracellular ROS levels induced by tau aggregate treatment, and at 50 μM, melatonin reduced phospho-tau as well as GSK3β mRNA and subsequent protein levels. Melatonin increased cell viability in tau-exposed neurons in a dose-dependent manner, with 80% viability observed at 20 μM melatonin and a complete reversal at 200 μM, compared to only a 60% viability in controls without melatonin . In an e fibrils , and thene (NAT) . Even thne (NAT)  by disrune (NAT) ,711 critne (NAT) , the conne (NAT)  or 5000 ne (NAT)  requiredne (NAT) ,708, or ne (NAT) . More imMLOs are found abundantly in the nucleus, cytoplasm, and on the membranes of almost all eukaryotic cells , where t−/−) mouse model demonstrated the formation of tau filaments elevated intraneuronal unesterified cholesterol, which may result in a vicious circle where tau fibrils alter cholesterol homeostasis and disturb cholesterol metabolism which continues to promote tau pathology [c domains [c domains [c domains to become more fluid, mechanistically deforming membrane bilayer structure [c domains that have become more fluid will transition into Lo lipid raft domains [Mutations in the Niemann–Pick type C (NPC) gene cause disturbances in cholesterol metabolism in lipid rafts , where aathology . Physiolathology . A recenathology . The binathology  includinathology . As a re domains  in anion domains . Increas domains  may caustructure . Melatontructure , bindingtructure . Lc doma domains ,724 that domains ,198.o–Ld phase separation over a range of temperatures [Melatonin may regulate lipid dynamics and composition, modifying the lipid hydrocarbon chain order to promote phase separation in ternary membrane models ,319, as eratures . The abieratures ; therefoeratures  can poteeratures ,722,725.The tumor suppressor p53 has been found to interact with tau and Aβ, forming pathological aggregates that result in the mislocation and impairment of its essential physiological DNA repair functions ,727,728.Often called the “guardian of the genome” , p53 is N-termini that flank the DBD [N-termini of p53 [More than 50% of all human cancers have been associated with missense  and/or s the DBD ,767,768. the DBD ; therefo the DBD . In addi the DBD ,771,772  the DBD . More imi of p53 .N- and C-termini disordered domains [Recent in vitro studies have demonstrated that formation of highly fluid p53 droplets at neutral and slightly acidic pH, and a low-salt environment is mediated by its  domains . The DNA domains . Molecul domains ,609. It A recent study demonstrated that wild-type p53 expressed in living yeast not only had the capacity to form liquid-like, dynamic, unstable droplets that appeared and disappeared in response to the presence or absence of stress, respectively, but when overexpressed, was able to propagate into true amyloid-like prions that could seed other molecules, and at the same time suppressed p53 transcription activities to precipitate tumorigenesis , confirmIn AD human brain tissues and animal models, the interaction between p53, pathological tau oligomers, and Aβ form aggregates resulting in the mislocalization and impairment of its important physiological functions in DNA repair ,727,728.The main function of UPS is to degrade and eliminate abnormal proteins damaged by oxidative stress and/or mutations after they are covalently bound to ubiquitin in an ATP-dependent pathway . Many MLIn eukaryotes, the UPS may be the most complex, extensive, cytosolic proteolytic enzyme system that performs essential functions  includin+, CoA into acetyl-coA, NADH and CO2. The phosphorylation of the E1α subunit of pyruvate dehydrogenase complex (PDC) by pyruvate dehydrogenase kinase 2 (PDK2) blocks the entrance of acetyl-coA into the tricarboxylic acid (TCA) cycle, inhibiting the OXPHOS production of higher ATP [The theoretical maximum of ATP calculated from simultaneous measurements of extracellular acidification and oxygen consumption indicated that OXPHOS ATP production was close to or more than 16 times above glycolysis, at 31.45 ATP/glucose  and 2 ATP/glucose, respectively . Whethergher ATP ,825,826.gher ATP ; thus, agher ATP . Decreasgher ATP  as well gher ATP .VDAC is the gatekeeper that controls the export of ATP out of mitochondria into cytosol and the import of essential respiratory substrates such as ADP and Pi into mitochondria ,399; the+, CoA into acetyl-coA, NADH and CO2, supporting the TCA cycle in OXPHOS to generate higher levels of ATP [p53 is a transcription factor  that ress of ATP ,825. AD s of ATP ,830; thes of ATP ,846,847 s of ATP ,851,852,s of ATP .Breast cancer (MCF-7) and human colorectal carcinoma cells (HCT116) treated with 1 μM melatonin exhibited p53 activation and accumulation which inhibited proliferation and protected against DNA damage through the ATP-dependent phosphorylation of p53 at serine 15 (Ser15) . PhosphoAs part of the complex PTM/UPS modification system, SUMO modification, similarly to ubiquitination, is an ATP-dependent enzymatic cascade involving activating, conjugating, and ligating E1, E2, and E3 enzymes, respectively ,873. SmaNucleoli PMLs are phase-separated quality control MLOs that compartmentalize misfolded proteins for clearance . In mammIn the cytoplasm, exposure to heat ,909, oxiFrontal cortex tissues of double-transgenic APP/PS1 AD mice that were given daily intraperitoneal injections of melatonin at pharmacological concentration of ~10 mg/kg  for 3 weeks exhibited a significant degradation of Aβ as a result of the SUMOylation of the amyloid precursor protein (APP) intracellular domain (AICD) at lysine 43 by the SUMO E3 ligase protein inhibitor of activated STAT1 (PIAS1) . AICD SU2O2 increased SUMO conjugation, but exposure to low levels of H2O2 (1 mM) induced a severe, rapid deSUMOylation within 5 min, resulting in the disappearance of SUMO conjugates including transcription factors [Stress granules are formed in response to external stress factors; therefore, hypoxia, heat, oxidative, osmotic, and genotoxic stress can also significantly increase SUMO conjugate levels as a protective response . Under s factors . Even th factors , a dysre factors  due to t factors  but also factors .Ribonucleic acid (RNA) is a single-stranded molecule with alternating ribose and phosphate groups attached to adenine, uracil, cytosine or guanine bases. The evolution of RNA is believed to precede that of DNA; nonetheless, the origin of the “RNA World” has not been resolved to date ,927,928.Phase-separated MLOs are enriched in RNA and RNA-binding proteins with IDRs ,630,941.Transcription factors (TFs) can undergo LLPS to form dynamic regions that compartmentalize and concentrate other TFs, enriching transcription-related proteins to activate the transcription of target genes . RNA proAberrant RNA–RNA interactions leading to the sequestration and/or dysregulation of RNA-binding proteins in MLOs may be one of the major driving forces behind neurodegenerative diseases ,948,949.6A) in the 5′ UTRs of mRNA [6A mRNA modification is dynamic, reversible, and has been observed to be oxidative-stress-dependent. Stress-induced methylation is recognized by the m6A cytoplasmic “reader” protein, YTH domain family 3 (YTHDF3) [6A to affect heterogeneous RNA and protein contents of SGs resulting from stress-specific differentiation in composition, dynamics of assembly and disassembly may ultimately determine the viability or pathology of cells in neurodegenerative diseases [The mechanism of selection for mRNA inclusion in SGs is determined by mRNA modification mediated by a prevalent methylation at position 6 of adenosine (m of mRNA . m6A mRN(YTHDF3) , which t(YTHDF3) . The abidiseases .6-methyladenosine (m6A), which involves the transfer of a methyl group to the sixth position of the purine ring in RNA adenosine [6A is installed by m6A methyltransferases and removed by m6A demethylases. RNA splicing, transcription, stability, and metabolism are all regulated by RNA m6A modifications [6A mediates structural switches that affect RNA stability and activity, regulating the access of RNA-binding proteins to their RNA binding motifs [6A is possibly the most prevalent internal, dynamic, reversible chemical modification identified to date, and plays critical roles in the growth, differentiation, and metabolism of cells [6A RNA methylation is involved in most aspects of RNA processing that may affect the regulation of cellular processes [Modification of eukaryotic messenger RNAs (mRNAs) occurs mostly via Ndenosine . m6A is ications ,955,956.g motifs . Among hg motifs , m6A is of cells ,959. An rocesses  such as rocesses ,962,963,rocesses , circadirocesses , fertilirocesses ,968,969,rocesses .6A RNA modification of eukaryotic RNAs is dynamic and reversible, where the methylation of mRNAs, tRNAs, rRNAs, and long non-coding RNAs by “writers” (RNA methyltransferases) such as METTL3 [6A-binding proteins) such as YTH domain proteins [6A “writers”, “readers”, and “erasers” are increasingly associated with degenerative and metabolic diseases. Fat mass and obesity-associated (FTO) protein, the m6A “eraser” associated with human obesity and energy homeostasis [6A “eraser”, was found to be overexpressed in the tumorigenesis of glioblastoma stem-like cells [6A “writer” was also identified as a critical regulator of a chromatin-based pathway that maintained cells in a leukemic state, where the inhibition of METTL3 removed the myeloid differentiation block in human and mouse AML cells [6A “reader”, was able to enhance antitumor immune responses in the dendritic cells of tumor-bearing mice [6A is increasingly associated with tumorigenesis [The ms METTL3  is removs METTL3 ,973, andproteins ,975. Dyseostasis ,978,979,eostasis , hepatoceostasis , melanomeostasis , and acueostasis ; howevereostasis . ALKBH5,ke cells . The METML cells . Depletiing mice . The dysigenesis ,988,989 igenesis ,992,993.6A methylation may be highest in the brain, regulating embryonic stem cell differentiation and brain development [6A methylation potentially drives neurodevelopmental disorders [6A RNA methylation in the brains of double-transgenic APP/PS1 with those of control mice revealed statistically significant elevations of m6A methyltransferase METTL3 and downregulations of m6A demethylase FTO in the cortex and hippocampus of AD mice [6A methyltransferases where METTL3 and the RNA-binding motif protein 15B (RBM15B) were downregulated and upregulated in the hippocampus, respectively. METTL3 was observed to be accumulated in the insoluble fractions of tau proteins, possibly implying an epitranscriptomic mechanism in altered gene expression in neurodegenerative disorders [melopment ,998,999;isorders . Investi AD mice . Post-moisorders . RNA epiisorders  may provisorders  to facilisorders .6A modifiers can lead to changes in the regulation of gene expression, affecting cancer [6A methylations, such as METTL3, METTL14, YTHDF1, YTHDF2, FTO, and ALKBH5, are significantly correlated with p53 mutations in AML patients [6A regulatory genes. In addition, their overall and event-free survival is worse than patients without m6A genetic alterations [6A methyltransferase METTL3 in hepatocellular carcinoma cells (HepG2) caused alterations in gene expression and alternative splicing in more than 20 genes, including MDM2, MDM4, and p21, involved in the signaling of p53 [6A methylation by METTL3 at the point-mutated codon 273 (G > A) of p53 pre-mRNA promoted a preferential pre-mRNA splicing that produced p53 R273H mutant genes that were resistant to multiple anticancer drugs in colon cancer cells [Dysregulation of mg cancer ,1004, neg cancer ,991,1005g cancer , blood pg cancer , and carg cancer . More thg cancer  and/or sg cancer ,760,761.patients . Most  inhibited SG formation and the recruitment of mRNAs into SGs [6A-RNA before they can be complexed into stress granules, implying that polymethylated m6A-RNA may act as a scaffold for YTH proteins, causing them to undergo LLPS through interactions within their own low-complexity domains [6A can be regarded as a beacon that attracts YTH proteins into stress granules [6A modifications.Aberrant RNA–RNA interactions leading to the sequestration and/or dysregulation of RNA-binding proteins in MLOs may be one of the major driving forces behind neurodegenerative diseases ,948,949.” YTHDF3 ,951. The” YTHDF3  enrichedinto SGs . YTH pro domains ,1018. Thgranules . PTMs sugranules  and SUMOgranules ,916. Mel6A “writers” and “readers” is also associated with the progression of cancer.Even though AICD SUMOylation has been shown to exert beneficial effects in transgenic AD mice models, the study of SUMOylation in neurodegenerative disorders and other diseases has led to controversial and often contradictory observations, because pathways can undergo SUMOylation at different sites, yielding conflicting consequences. For example, the SUMOylation of many important proteins in AD, including APP and tau, have been associated with the pathogenesis of AD ,1021 and6A levels in vitro and in vivo—a process that can be reduced by deSUMOylation enzyme sentrin/SUMO-specific protease 1 (SENP1) [6A “reader” YTHDF2 by SUMO1 at lysine residue K571 in vitro and in vivo increased the binding affinity of YTHDF2 to m6A-modified mRNAs, altering gene expression profiles, resulting in the increased proliferation, migration, colony formation and tumor growth of lung cancer H1299 cells [The SUMOylation of METTL3 was found to promote tumor growth and colony formation in human non-small cell lung carcinoma (NSCLC) cells (H1299) , where t (SENP1) ,1040. In (SENP1) . However99 cells .6A “reader” targets and destabilizes m6A-modified mRNAs, facilitating the localization and degradation of m6A mRNA in MLOs such as P-bodies [5C, a ubiquitous post-translational RNA modification found in mRNAs [5C in RNA, significantly regulating 208 out of 1350 identified m5C sites that may affect pre-rRNA processing through the modification of m5C levels in rRNA [The YTHDF2 mP-bodies ,953. UltP-bodies . The proin mRNAs . YTHDF2  in rRNA . More imN-acetyl-l-cysteine (NAC), an antioxidant, also augments YTHDF2 expression in oxidative-stress-induced senescent cells; therefore, it is believed that oxidative pathways may negatively regulate YTHDF2 expression [Stimulation of oncogene Ras led to the suppression of YTHDF2 that stabilized the transcription of MAP2K4 and MAP4K4, upregulating the senescence-associated secretory phenotype (SASP) in human ovarian surface epithelial cells (HOSEpiC). Treating HOSEpiC cells with 1 μM of melatonin enhanced the expression of YTHDF2, reversed telomere shortening, and blocked Ras-induced growth arrest . The actpression . On the Adult male C57BL/6J mice pretreated with melatonin intraperitoneal injections (25 mg/kg b.w./day × 14 days) exhibited attenuated cell viability loss, ROS generation, mitochondrial dynamics imbalance, and mitophagy in spermatogonial stem cells (SSCs) induced by daily intraperitoneal injections with chromium (VI) (16.2 mg/kg b.w./day × 14 days), an environmental toxin and carcinogen that can cause male infertility by damaging SSCs ,1048. In6A regulates the pluripotency of embryonic stem cells (ESCs) [6A modification and significant reduction in m6A “writer” METTL3 [6A mRNA methylation and altered the subcellular location of METTL3, preventing m6A-dependent mRNA decay to stabilize key pluripotency factors Nanog, Sox2, Klf4, and c-Myc [6A methylation [6A modification compared to wild-type ESCs treated with melatonin [6A mRNA methylation.On the other hand, melatonin was found to decrease METTL3 expression and modification in another report. ms (ESCs) . Treatme” METTL3 . Melatonnd c-Myc , known thylation . It has hylation . Using delatonin . Howeverelatonin ) and/or elatonin ) loweredelatonin , then it6A mRNA methylation can be oxidatively reversed or “erased” by m6A demethylases such as FTO [mh as FTO ,1055 andh as FTO ,1056. Juh as FTO , the defh as FTO . In facth as FTO . DDX3 ish as FTO . DDX3 wah as FTO .NLRP3 inflammasome, a widely documented target of melatonin associated with pathological protein aggregates in neurodegenerative disorders ,313,314,When life originated, LLPS driven by multivalent macromolecular interactions might have been the organizing principle behind the subcellular compartmentalization of MLOS in eukaryotes and prokaryotes ,277,428.Adenosine triphosphate (ATP) is one of the four nucleotide monomers used during RNA synthesis . RNA hasCells rely upon RNA to regulate condensates because RNA molecules contain powerful electrostatic forces due to the high negative charge densities buried in their phosphate backbones ,531,532.Saccharomyces cerevisiae in 1999, DX19 (human)/Dbp5 (yeast) [Since its discovery in  (yeast)  has been (yeast) ,1081, mR (yeast) , and tra (yeast) , as well (yeast) ,1084. On (yeast) . NPCs no (yeast) .It has been proposed that the ATP-dependent catalytic cycle of DDX19 involves the cycling between open and closed conformations to bind RNA for export into cytoplasm . Even thNPCs can be visualized as thousands of toroid-shaped “ultradonut”-like pores with extremely high curvatures generated by nanoscale buckling instabilities triggered by membrane stresses during nuclei growth . These “Saccharomyces cerevisiae with defects in regulating membrane fluidity assembled NPCs that were defective, whereas the restoration of membrane fluidity via the addition of membrane-fluidizing agents attenuated defects in NPC biogenesis and normalized mRNA export [•OH [•OOH, the two most prevalent ROS responsible for the chain oxidation of unsaturated phospholipids [As early as 1979, the regulation of nuclear RNA release was found to be directly correlated with nuclear membrane fluidity where a reduction in membrane fluidity caused a linear decrease in RNA release . It has A export . The perA export ,1101 mayA export ,354,355.A export ,341,356.A export , especiaort [•OH  and •OOHholipids ,466 suchholipids  , preserving nanoscopic lipid domain structure and composition, possibly by reducing membrane line tension [o-phase lipid rafts in cells and nuclei (During protein membrane adsorption, the complex interactions between lateral membrane organization and proteins often enhance the propensity of membrane lipids to form domains or to phase-separate . These d binding . Cells m tension . Lipid p tension ,218,296.d nuclei .Rhodospirillum rubrum and cyanobacteria, precursors to mitochondria and chloroplasts, respectively [The relationship between melatonin and RNA is likely an ancient one that might date as far back as ~4 billion years ago, possibly after the height of the “RNA world” ,1117, whectively ,424,425,ectively ,426,427.The physiological and pathological functions of biomolecular condensates in neurodegenerative disorders are shaped by powerful, complex, interdependent relationships between membraneless organelles, membranes/lipid rafts, ATP, RNA, and most of all, stress and its timely resolution. Melatonin’s intimate association with each of these decisive influencers may position the potent, ancient antioxidant as an important mediator of the phase separation of condensates in health and disease via principal ATP-dependent mechanisms including post-translational modifications and RNA m6A modifications . This no


Metabolite genome-wide association studies (mGWAS) are key for understanding the genetic regulation of metabolites in complex diseases including cancers. Although mGWAS has revealed hundreds of metabolomics quantitative trait loci (mQTLs) in the general population, data relating to gastric cancer (GC) are still incomplete.We identified mQTLs associated with GC by analyzing genome-wide and metabolome-wide datasets generated from 233 GC patients and 233 healthy controls.Twenty-two metabolites were statistically different between GC cases and healthy controls, and all of them were associated with the risk of gastric cancer. mGWAS analyses further revealed that 9 single nucleotide polymorphisms (SNPs) were significantly associated with 3 metabolites. Of these 9 SNPs, 6 loci were never reported in the previous mGWAS studies. Surprisingly, 4 of 9 SNPs were significantly enriched in genes involved in the T cell receptor signaling pathway.Our study unveiled several novel GC metabolite and genetic biomarkers, which may be implicated in the prevention and diagnosis of gastric cancer. Gastric cancer (GC) is one of the most common, multifactorial malignancies mediated by environmental and genetic factors , 2. SeveGWAS with intermediate phenotypes, like changes in metabolite and protein levels, are key in establishing functional links between genetic variants and disease end points . MetabolGWAS for metabolic traits  has revealed hundreds of metabolomic quantitative trait loci (mQTLs) in the general population , 13–15. H. pylori infection-, and time of blood sample collection-matched to healthy controls. We hypothesized that by combining phenotypic, metabolomic, and genetic data, we could better identify individuals at risk of GC and uncover biological mechanisms related to GC.To identify the GC-associated mQTLs for the first time, we combined genome-wide and metabolome-wide datasets generated from 233 GC patients and 233 healthy controls. Patients were age-, sex-, smoking status-, alcohol consumption-, This study was based on a population-based case-control study, in which GC patients and healthy individuals were consecutively enrolled in Xianyou County between March 2013 and December 2017. All cases were newly diagnosed histologically based on tissue specimens and had lived in Xianyou for at least 10 years. GC patients with other cancers, secondary or recurrent gastric cancer, gastritis, previously received neoadjuvant chemotherapy or chemoradiotherapy or radiotherapy, pregnant, metabolic diseases such as diabetes, gout, hyperlipidemia, systemic administration of corticosteroids, neurological and psychiatric diseases, severe hepatic and renal dysfunction, and severe respiratory disease requiring continuous oxygen treatment, etc., were excluded. For the healthy individuals, there was no abnormality of blood tests and endoscopic examinations.The study initially included 244 GC cases and 244 healthy individuals. Ethical approval for the study was obtained from the Biomedical Research Ethics Committee of Fujian Medical University, China . Following an explanation of the study, written informed consent was obtained from all participants at study enrollment.Blood samples were collected after an overnight fast, immediately centrifuged, and stored at −80°C until assayed. Plasma metabolites were profiled as previously described using Agilent 1200 high-performance liquid chromatography combined with a 6520 accurate electrospray ionization/quadrupole-time-of-flight mass system  . A totalp < 1 × 10−6). As a result, 233 pairs of subjects and 258,544 SNPs were left for subsequent analysis.Approximately 900,000 SNPs were genotyped using the Axiom™ Precision Medicine Research Array . Genotyping was performed as described according to the manufacturer’s instructions. Briefly, genomic DNA was extracted using a Genomic DNA Isolation Kit . Each sample was whole-genome amplified, fragmented, precipitated, dried, resuspended in appropriate hybridization buffer, chip cleaned, stained, and scanned. Participants were excluded if they satisfy any of the following items: (1) low call rate , (2) ambiguous gender, and (3) duplicates or familial relationship (PI_HAT > 0.025). SNPs were excluded if they (1) were not mapped to autosome chromosomes, (2) had a call rate < 95%, (3) had minor allele frequency (MAF) < 0.05 in controls, and (4) were an excessive deviation from Hardy-Weinberg equilibrium in controls  method was used to correct for multiple hypothesis testing and to reduce false positives. Those metabolic features with variable importance in the projection (VIP) values > 1.0 in the OPLS-DA model and FDR-adjusted p values < 0.05 in the t-test were considered to be significantly different between GC cases and healthy controls. Then, logistic regression was performed to test the association between discriminant metabolites and incident GC.Before statistical analysis, each metabolic peak in all subject samples was normalized based on QC samples for removing the unwanted analytical variations occurring intra- and inter-batches. And the plasma abundant values of metabolites investigated were set to a log scale and auto-scaled (mean-centered and divided by the standard deviation of each variable) using MetaboAnalyst 4.0. An orthogonal partial least squares discriminant analysis (OPLS-DA) and univariate two-sides H. pylori infection analysis in TASSEL software (version 5.0). To control for false-positive error rates deriving from the large number of SNPs tested, a conservative Bonferroni-adjusted p-value of p = 8.79 × 10−9 (= 0.05/(258544SNPs × 22 metabolites)) was applied for declaring genome-wide significance for the SNP-metabolite associations. The significant SNPs were annotated to the neighboring genes of 1000 Genomes Project (hg19/1000 Genomes ASN) downloaded from the University of California Santa Cruz (UCSC) genome browser (http://hgdownload.cse.ucsc.edu). Then, we conducted Gene Ontology (GO) enrichment analysis to investigate possible biological, molecular, or cellular processes associated with significant SNP-related genes using Metascape (https://metascape.org/) [https://www.cancer.gov/tcga), and t test was performed to compare the candidate gene expression between two different groups shown in boxplot using R (R version 3.6.0).After that, we analyzed the associations between discriminant metabolite levels and genome chip variants using a generalized linear model adjusted for age, sex, family relationship, smoking status, alcohol consumption, pickled vegetable intake, and pe.org/) . Furtherp < 0.05), there was no statistically significant difference in gender, age, smoking status, alcohol consumption, and H. pylori infection, between GC cases and healthy controls (p > 0.05).A total of 233 GC cases and 233 healthy controls were included, with the mean age of 64.80 ± 7.89 years and 65.16 ± 7.91 years, respectively. As shown in Table R2Y) and prediction ability of the model (Q2) were 0.472 and 0.420 for differentiating GC cases and healthy controls, respectively, suggesting successful model construction.To identify potential compounds that could be used to differentiate the metabolite profiles of GC cases and healthy controls, we established a supervised OPLS-DA model that focused on the actual class discriminating variation. As shown in Fig. t-test and the VIP scores from OPLS-DA models were carried out. Metabolites with FDR-adjusted p-values < 0.05 and VIP >1 were selected as discriminants. Figure Subsequently, to find discriminant metabolites between GC cases and healthy controls, a univariate two-sides l-palmitoylcarnitine, testosterone, dihydrobiopterin, and paraxanthine, were associated with increased risks of GC. Conversely, metabolites including guanosine triphosphate (GTP), Cer(d18:0/12:0), 8-Isoprostaglandin E1, platelet-activating factor, TG(22:5/15:0/22:5), SM(d18:1/16:0), cholic acid, indolelactic acid, indole-3-lactic acid, porphobilinogen, and l-histidinol were associated with decreased risk of GC. Receiver operating characteristic (ROC) curve analysis was used to assess the discriminative ability of the 22 metabolites. The area under the curve was 0.897 (95% CI 0.869 to 0.924), indicating a high diagnostic value for GC. In short, these metabolites are highly correlated with the occurrence of GC.Figure −9. Of these, 4 loci are in the enhancer regions, 2 are in the 3′UTR. Besides, 6 loci have not been reported before, while 3 loci were reported in the previous mGWAS study in the general population [r2 = 1) with the variant rs12202419. And markers R2 for 3 metabolites ranged from 7.23 to 8.46% and the total phenotypic variance explained by 9 SNPs was 70.46%.Next, we analyzed the association of these 22 metabolites with genome chip variants. As listed in Table pulation , includiPDE4D, BTN3A3, BTN2A2, BTN3A1, BTN2A3P)  with five genes , which indirectly verifies the reliability of this study.Finally, GWAS was performed by plink to investigate whether the genotype distribution of the 9 SNPs is different between GC cases and healthy controls. Unfortunately, no statistically significant association was found. Then, we downloaded RNA-seq data of gastric cancer tissue and adjacent normal tissue from TCGA and compare the expression of 10 candidate genes between GC tissue and adjacent normal tissue. As shown in Fig. In this study, 22 metabolites were found to be associated with the occurrence of GC, including 7 nucleotides, 9 lipids, 5 organoheterocyclic compounds, and 1 amino acid.Nucleotide synthesis is key for cell proliferation as it is required for DNA replication, gene transcription, and ribosome biogenesis . Tumor cl-palmitoylcarnitine, testosterone, Cer(d18:0/12:0), 8-Isoprostaglandin E1, platelet-activating factor, TG (22:5/15:0/22:5), SM (d18:1/16:0), and cholic acid were found to be associated with GC. Yu et al. demonstrated that there was no difference in plasma linoleic acid concentration between gastric cancer patients and healthy individuals [l-palmitoylcarnitine levels were found in mice with colon cancer and patients with acute pneumonia, indicating an increase in inflammation and fatty acid beta-oxidation [l-palmitoylcarnitine may also play a pro-inflammatory role in gastric cancer.Lipids, including phospholipids, fatty acids, triglycerides, sphingolipids, cholesterol, and cholesteryl esters, are key constituents of all biological membrane structures , 28. Morividuals , but somividuals , 33. Linividuals . Higher xidation , 36. FinCer(d18:0/12:0) and SM(d18:1/16:0) are members of sphingolipids (SPs), which are structural molecules of cell membranes with important roles in maintaining barrier function and fluidity , 37. SphPlatelet-activating factor, also known as PAF or PC(O-16:0/2:0), is a ubiquitous, potent phospholipid activator and mediator of inflammation that plays an important role in the pathogenesis of inflammatory disorders . In vitrThe relative abundance of dihydrobiopterin (BH2), the precursor of tetrahydrobiopterin (BH4) of folate biosynthesis, was increased in gastric cancer patients compared with the healthy controls. Tetrahydrobiopterin (BH4) is essential for the synthesis of nitric oxide (NO) , and it 95% CI 1.12 to 1.62), indicative of an ideal intermediate phenotype choice of GC. Therefore, we proceeded to identify mQTLs associated with GC by testing the genetic associations of 22 discriminant metabolites with genome-wide data. We found that 9 SNPs were significantly associated with 3 metabolites. Of them, 3 loci were reported in the previous mGWAS [Although we found some metabolites associated with GC, the biological mechanisms remain unclear. Hence, it is essential to combine metabolomics with other -omics methods to get a more integrated understanding of gastric carcinogenesis. mGWAS is an excellent method in quantifying metabolic data and uncovering genetic variants affecting metabolite levels . In thisus mGWAS , and 6 lBesides, 6 SNPs were found to be associated with PAF, which influences the occurrence of gastric cancer. They included rs12208390, rs3927423, and rs6912853 in butyrophilins (BTN) gene; rs140044870 in DCLK2; rs12204145 in ABT1; and rs957788 in FAM155A. The BTN gene belongs to the immunoglobulin superfamily. There are seven human BTN genes in three related phylogenetic subfamilies. BTN1 subfamily contains only the BTN1A1 gene, while BTN2 contains BTN2A1, BTN2A2, and BTN2A3 pseudogenes, and BTN3 contains BTN3A1, BTN3A2, and BTN3A3 genes . StudiesPrevious studies revealed that the Neuronal tyrosine-phosphorylated Adaptor for The PI3-kinase (NYAPs) could activate the PI3K signaling pathway . Moreove95% CI 0.869 to 0.924), indicating a high diagnostic value for GC and an ideal intermediate phenotype choice of GC. More importantly, we identified 6 new mQTLs in GC cases that may be a valuable tool for discovering genetic biomarkers related to GC. Therefore, this paper provides new insights into the prevention, early detection, diagnosis, and targeted treatment of gastric cancer. However, a validation cohort and in vivo and in vitro experiments are required to confirm these findings and fully explore the implications of this study.In summary, we found 22 metabolites that were statistically different between GC cases and healthy controls. All of them were associated with the risk of gastric cancer. The area under the ROC of 22 discriminant metabolites was 0.897 (