Mycobacterium spp., Listeria spp., Chlamydia spp., Mycoplasma spp., Rickettsia spp., Legionella spp. and Yersinia spp., which can modulate and reprogram of defense genes in host innate immune cells.Some intracellular pathogens are able to avoid the defense mechanisms contributing to host epigenetic modifications. These changes trigger alterations tothe chromatin structure and on the transcriptional level of genes involved in the pathogenesis of many bacterial diseases. In this way, pathogens manipulate the host cell for their own survival. The better understanding of epigenetic consequences in bacterial infection may open the door for designing new vaccine approaches and therapeutic implications. This article characterizes selected intracellular bacterial pathogens, including  Helicobacter pylori [Porphyromonas gingivalis, Fusobacterium nucleatum [Streptococcus bovis, Chlamydia pneumoniae, Campylobacter rectus, Epstein–Barr virus, hepatitis viruses, human papilloma virus, polyoma viruses, can contribute to the host epigenetic changes and are frequently associated with carcinogenesis [Mycobacterium tuberculosis—an obligatory, aerobic bacillus still remaining one of the major global health problems since it is estimated that one fourth of global human population is infected with that pathogen [Listeria monocytogenes—mainly transmitted through the consumption of contaminated food, causing listeriosis, a disease whose importance is not sufficiently recognized [Chlamydia, Mycoplasma, Rickettsia, Yersinia and Legionella [Epigenetic regulation of the gene activity is a subject of deep and still-increasing interest. The appearance of an epigenetic trait defined as “a stably heritable phenotype resulting from changes in a chromosome without alterations in the DNA sequence” can be triggered by changes in the environment of the cell, precisely described and discussed by Berger et al. . There ir pylori , Porphyrucleatum , Streptoogenesis . In the ogenesis )—a factoogenesis ,6,7. Howogenesis . In thispathogen ,10 and Lcognized . We havegionella ,13,14. TMycobacterium tuberculosis—the causative agent of tuberculosis (TB) in humans—is equipped with a broad spectrum of tools that allow it to survive and avoid the defense mechanisms of the host [M. tuberculosis, M. bovis), while the other ones (M. smegmatis) do not express Rv1988. Following the tubercle bacilli entering the cell, released Rv1988 localizes with the chromatin in the host nucleus affecting the expression of genes, which are important for host defense, e.g., NOX1, NOX4, NOS2 and TRAF3 [M. tuberculosis shows an ability to localize to the nucleus inside the infected mammalian cell. Rv2966c binds to specific DNA sequences and causes predominantly non-CpG methylation, and its activity is positively influenced by phosphorylation [M. tuberculosis strains) of blood monocyte-derived macrophages, but not THP-1 cells, the enhancement in the hyper-methylation level occurred in the case of genes encoding IL17RA, IL15RA, IL6R and IL6ST, whereas the IL4R methylation was intensified in both types of macrophages [M. tuberculosis strains (Beijing/W and non-Beijing/W strains), the significantly higher hypermethylation levels of the IL17RA, IL15RA, IL4R, IL6R, IL6ST genes as well asIL17RA, IL6R, IL6ST, respectively, were observed, when compared with uninfected macrophages [M. tuberculosis—or exposed to Rv3763—the IFN-γ increased acetylation of histones H3 and H4 at CIITA pIV locus was inhibited. It was also shown, that 19-kDa lipoprotein required TLR2 to mediate its inhibitory effect on IFN-γ-dependent chromatin remodeling of MHC2TA (the gene encoding CIITA). Furthermore, when macrophages were treated with inhibitors of MAPKs p38 or ERK, the inhibition of CIITA expression or IFN-γ-dependent histone acetylation of CIITA pIV by 19-kDa lipoprotein was not observed. Additionally, Rv3763 also inhibited IFN-γ-dependent recruitment of the ATP-dependent chromatin-remodeling protein, Brg1, to CIITA pIV. All of this suggests that M. tuberculosis-mediated disruption of IFN-γ-dependent chromatin rearrangement at MHC2TA leads to the inhibition of CIITA transcriptional activity [M. tuberculosis strains in the context of 22 genes involved in the TLR2 signaling pathway [Irak-2 and Tbk-1, were characterized by increased methylation levels when THP-1 macrophages were infected with XDR M. tuberculosis strains. In the case of infection with the susceptible strains, besides the increased methylation level of Traf6 gene and hypomethylation of Irak-2 gene, normal methylation of the other tested genes related to the TLR signaling pathway was observed, which may induce effective responses to infection. It was also shown that in all used drug-resistant mycobacterial strains the expression of Rv1988 gene was elevated in comparison with susceptible strains and that was accompanied by the THP-1 methylation status in response to infection with those two groups of mycobacterial strains [Mycobacterium tuberculosis, a stable demethylation at enhancer elements was induced, including those associated with the H3K4me1 enhancer mark. It was found that immune-related transcription factors (TFs) such as NF-κB/Rel were recruited to enhancer elements before the observed losses in methylation, which suggests that DNA demethylation is mediated by TF binding to cis-acting elements [CREB5, REL, NFKB1, IRF2, IRF4, as well as CD83 and BCL2 [the host ,16,17. Othe host . Yaseen the host  describend TRAF3 . The firnd TRAF3 ,21 and tnd TRAF3 ,23,24. Irylation . Similarrylation . As the rylation . It is brylation ,27. Usinrophages . Ip et arophages , which hrophages . It turnactivity . Interes pathway . All of  strains . Apart f strains ,34. The elements . Pacis eelements  indicateand BCL2 .Listeria spp. is another interesting example of microorganism that can induce epigenetic and miRNA modifications in the host to modulate immune defense. Listeria monocytogenes is a Gram-positive facultative pathogen that causes the foodborne disease—listeriosis [L. monocytogenes is internalized into a vacuole. To escape from this niche by physically disrupting the vacuolar membrane it uses listeriolysin O (LLO) and phospholipase A and B (PlcA and PlcB) [L. monocytogenes is able to survive and replicate in the cytosol of host cells and modify host cell processes as well as organelles [L. monocytogenes involves actin assembly inducing protein (ActA), which interacts with the Arp2/3 (actin-related protein 2/3) complex and mediates actin polymerization generating an actin comet tail.This allows bacteria to spread from one cell to another [L. monocytogenes manipulate host cell transcription and induce epigenetic modifications? One of the important classes of bacterial virulence factors are nucleomodulins that are secreted into the host cytoplasm and migrate to the nucleus to modulate gene expression. One of them is Listeria nuclear targeted protein A (LntA), which interacts with the chromatin repressing bromo adjacent homology domain-containing oneprotein (BAHD1) resulting in the upregulation of interferon-stimulated genes (ISG) in a type III interferon-dependent manner [L. monocytogenes also secrete cyclic small dinucleotide called cyclic di-AMP (c-di-AMP), which activates the stimulator of interferon genes protein (STING). The activation of innate immune pathways affects the stimulation of T cells resulting in the impaired clearance of the pathogen [teriosis ,38. Follnd PlcB) ,40. In gganelles . The cel another ,42. How t manner . Moreovepathogen ,44. ThesL. monocytogenes induced NF-κB signaling and p38 MAPK (mitogen-activated protein kinases) phosphorylation via Nod 1. Moreover, they revealed that activation of Nod1was not essential for IFN-γ production, but it was crucial for IL-8 response [The host cytoplasm contains nucleotide-binding oligomerization domain (Nod) proteins, which detect peptidoglycan in the bacterial cell wall. Nod is involved in the induction of the nuclear factor κB (NF-κB) activity, including activation of the receptor-interacting protein 2 (Rip2) and IκB kinase, as well as in the activation of caspases ,46. A stL. monocytogenes [As a key virulence factor, LLO has an ability not only to form pores in the cell membrane, but also to induce MAPK as well as calcium and NF-κB signaling. Moreover, LLO induces dephosphorylation of Ser10 on histones H3 and deacetylation of histones H4 ,48. Histytogenes ,49.L. monocytogenes infection. miR-146a was found to regulate the activity of TNF receptor-associated factor 6 (TRAF6), IL-1 receptor-associated kinase 1 and 2 (IRAK1 and IRAK2), whereas miR-155 influenced IκB kinase epsilon (IKKε) [hly mutant strain did not abolish the activity of miRNAs [L. monocytogenes inhibited IFN-γ-induced autophagy of macrophages viamTOR  by Mir155 and Mir31 in the WNT signaling network and the activity of protein phosphatase 2 (PP2A) [MicroRNAs (miRNAs) are key modulators that affect the outcome of immune responses to infection at post-transcriptional levels . Schnitgn (IKKε) . Interesf miRNAs . Moreove2 (PP2A) .Chlamydia trachomatis, Chlamydophila pneumoniae, Chlamydophila psittaci and Chlamydophila pecorum [C. trachomatis and C. pneumoniae, the best-known species, are a cause of sexually transmitted diseases, ocular infections and atypical pneumonia [Chlamydiae are obligate, intracellular, Gram-negative bacteria with a unique developmental cycle of replication consisting of extra- and intracellular forms. Within the family there are four species:  pecorum . They arneumonia ,56.C. trachomatis genome identified a set gene encoding a protein with a domain similar to the eukaryotic SET domain, which is known to methylate lysine residues in the amino-terminal tail of histones [C. trachomatis called NUE has been shown to be secreted into the host cell during chlamydial infection, where it enters the nucleus and binds to host chromatin.It has been found that pathogenic chlamydiae have evolved diverse strategies to suppress the host cell response, mainly by targeting chromatin regulation via epigenetic modifications . Analysihistones ,59. ThisC. pneumoniae was called cpnSET and its activity was determined against chlamydial histone-like proteins Hc1 and Hc2 as well as against mouse histones [As a methyltransferase, the NUE is able to catalyze methylation of host histones H2B, H3 and H4. The homolog of NUE in histones . Apart fhistones . It is sChlamydophila psittaci via a type III secretion system (T3SS) targets the inner membrane of the nucleus in infected cells and thus may control chromatin interaction with the nuclear lamina [Nucleomodulins may not only bind DNA and chromatin factors, but also chromatin-anchoring factors. SinC, a protein secreted by r lamina .Mycoplasma hyorhinis, an intracellular commensal that can shift to an opportunist pathogen. The Mhy1 and Mhy2methyltransferases, promote CG methylation, while the Mhy3 enzyme acts on GATC sites [M. hyorhinis.Mycoplasmas are the smallest self-replicating Gram-negative bacteria, which lack the genes coding for the cell wall. Instead of a cell wall, they possess a three-layered membrane, containing sterol, which is taken up from the environment. In humans, mycoplasmas are present frequently at mucosal surfaces of respiratory and urogenital tracts, mammary glands and joints . Due to TC sites . ChernovTC sites  demonstrRickettsia spp. are obligate intracellular Gram-negative bacteria that require a vector for host transmission [smission ,66. Ricksmission . Rickettsmission ,68,69. Tsmission . The bacsmission ,72,73. Rsmission ,75. Ricksmission ,76.Intracellular pathogens have developed mechanisms that allow survival within hostile host environment. As a result of the bacterial influence on host chromatin modifications and defense gene transcription, intracellular pathogens cause dysregulation of host cell function leading to disease ,77. The R. conorii and R. montanensis. They analyzed transcriptomic changes in 495 host genes as early as 1 h post-infection. The differentially expressed genes included the pro-inflammatory molecules TNFα, IL1β, as well as CCL20, CCL3L3, CCL3, CCL4L2, CXCL1, CXCL3 and CXCL8 that could shape the recruitment of immunocompetent cells to the site of infection. Moreover, they noticed changes in genes implicated in the modulation of the NF-κB pathway, including tumor necrosis factor, alpha-induced protein 3 (TNFAIP3) and NF-κB inhibitor zeta (NFKBIZ) [R. conorii enhanced the expression of mRNA for proteins involved in the JAK-STAT  signaling pathway. Considerable changes were observed in the genes for IL23A (interleukin 23 subunit A), OSM (oncostatin M) and SOCS3  [R. conorii is able to modulate TNFα signaling in macrophages, which may influence inflammatory cell activation. In the study by Curto et al. [R. conorii infected cells responded with overproduction of TNFα following the treatment of THP-1 cells with LPS E. coli. This correlation was not observed in uninfected THP-1 cells or in the cells infected with non-pathogenic R. montanensis.Curto et al.  investig(NFKBIZ) . Both of(NFKBIZ) ,82,83. Ialing 3) . All of aling 3) . The genaling 3) . As a plaling 3) ,87,88. Io et al.  R. conorThe rickettsial species can also modulate immunometabolism, although this process is still not well understood. Recent proteomic studies have revealed that rickettsiae modify the activity of host proteins involved in various metabolic processes . SignifiRickettsiae-treated THP-1 culture there was observed overproduction of citrate synthase, aconitase, isocitrate dehydrogenase 3 (IDH3A), fumarate hydratase (FH) isocitrate dehydrogenase 1 (IDH1) as well as malate dehydrogenase 1 and 2 (MDH 1 and 2) [Rickettsial infection has been found to reduce the activity of enzymes involved in glycolysis and PPP, including glucose-6 phosphate isomerase, fructosebiphosphatase A, phosphoglycerate kinase 1, enolase 1, pyruvate kinase M1/2 and lactate dehydrogenase B . These p1 and 2) . All of 1 and 2) .Rickettsiae resulted in the accumulation of proteins involved in the electron transport chain including cytochrome C1 (CYC1), ubiquinol-cytochrome c reductase core protein 1 and 2 (UQCRC1 and 2), cytochrome c oxidase subunit 4l1 (COX4l1) and cytochrome c oxidase subunit II (COX2).Moreover, Curtoet al.  found thRickettsiae is associated with an increase in the expression of the host proteins responsible for lipid metabolic processes. The major role in this process is played by fatty acid synthase (FAS) involved in de novo fatty acid synthesis, which can be used in the life cycle of bacteria [Noteworthy is the fact that infection of THP-1 cells with bacteria ,92.The differences observed in the activity of enzymes involved in metabolic pathways suggest reprogramming of defense genes in macrophages, induced by the rickettsial species.Legionella spp., a facultative intracellular Gram-negative bacterium, is an etiologic agent of the atypical pneumonia called the Legionnaires’ disease as well as the flulike infection known as the Pontiac fever [Legionella pneumophila (L. pneumophila), responsible for most of the identified cases of legionellosis, is transmitted through inhalation of bacteria-containing aerosols from natural and artificial water sites [ac fever . The sevac fever . Legioneer sites .L. pneumophila interferes with a wide range of host processes using them for its intracellular replication and survival [L. pneumophila are able to ubiquitinate, phosphorylate, lipidate, glycosylate, AMPylate, de-AMPylate, phosphocholinate and dephosphocholinate various proteins of the host [survival . The patthe host .U-box domains (U-box1 and U-box2) with similarity to eukaryotic E3 ubiquitin ligases, which can target cellular Clk1 (Cdc2-like kinase 1) during L. pneumophila infection [U-box containing protein, L. pneumophila also secretes some F-box-containing proteins  that interfere with ubiquitin signaling during infection of host cells. AnkB protein (L. pneumophila strain A100) containing the CAAX motif, promotes bacterial intracellular replication by recruitment of polyubiquitinated proteins on the Legionella-containing vacuole, whereas AnkB of L. pneumophila strain Paris lacking the CAAX motif modulates the ubiquitination of ParvB, a host protein present on focal adhesions and in lamellipodia [L. pneumophila LegU1 effector conferring E3 ubiquitin ligase activity has been found to target the host chaperone protein BAT3, a regulator of the endoplasmic reticulum stress response [The pathogen modulates the ubiquitin signaling pathway by secreting molecules that mimic certain eukaryotic proteins. A LubX protein contains two nfection . Moreovenfection . Apart fresponse .L. pneumophila serine/threonine protein kinases are LegK1 and LegK2 [The process of protein phosphorylation plays a key role in the regulation of cell growth, differentiation or apoptosis. Two best-studied nd LegK2 . LegK1 pnd LegK2 . LegK2 knd LegK2 .L. pneumophila to promote its pathogenesis and survival. Three Legionella glycosyltransferases—Lgt1, Lgt2 and Lgt3—target eEF1A at Ser53 (eukaryotic translation elongation factor 1A), the most abundant protein synthesis factors and thereby inhibit the eukaryotic protein translation process [L. pneumophila glucosyltransferase SetA, introduced into target cells by a type IV secretion system, affects the intracellular host vesicle trafficking pathways [Host glycosylation regulating cell signaling or gene transcription is another strategy used by  process . An addipathways .L. pneumophila. AMPylation is a process in which an adenosine monophosphate (AMP) moiety is covalently added to threonine, tyrosine of serine residues of a protein by using ATP [L. pneumophila DrrA/SidM effector modulates adenylylation of Rab1b protein, the host regulatory protein recruited during the infection to the Legionella-containing vacuole [L. pneumophila protein AnkX transfers a phosphocholine group from CDP-choline to a serine in the Rab1 and Rab35 GTPases [Legionella Lem3 (lpg0696) effector possessing an activity opposite to that of AnkX can remove the phosphocholine moiety from Rab1 [AMPylation (adenylylation) and de-AMPylation, as well as phosphocholination and dephosphocholination, are other post-translational modifications of host proteins exploited by sing ATP ; L. pneu vacuole ,108,109. GTPases , whereasrom Rab1 .Yersinia pestis, a facultative intracellular Gram-negative bacterium, is an etiological agent of plaguethat can infect humans via the oriental rat flea [Yersinia effector proteins named Yops across the host immune cell membrane, where they can exert cytotoxic and immunomodulatory effects [rat flea . Thepathrat flea . One of  effects . Nucleom effects ,116,117. effects .Understanding the role of epigenetic reprogramming in the pathogenesis of infectious diseases is a challenging perspective. Elucidation of how chromatin remodeling influences the function of the innate immune system during the pathogen infection and following recovery, provides a scientific basis for arising new therapeutic opportunities. Recent data highlight a number of different pharmacological compounds used in vitro and in vivo that influence the immune response through macrophage tolerance and training. The development of site-specific histone deacetylase (HADC) inhibitors, bromodomain inhibitors, histone lysine methyltransferase (HKMT) inhibitors represent new tools in the modulation of histone post-translational modifications. The HDAC inhibitors were found to be a promising approach to purge the reservoir of persistent HIV infection , whereasEpigenetic reprogramming leading to the chromatin rearrangement and, in consequence, to alterations in the level of expression of certain factors/proteins is considered to be a crucial player determining the fate of cells/organisms. Epigenetic changes have been identified as the key events present in the course of the trained immunity phenomenon  as well


The global prevalence of metabolic disorders, such as obesity, diabetes and fatty liver disease, is dramatically increasing. Both genetic and environmental factors are well-known contributors to the development of these diseases and therefore, the study of epigenetics can provide additional mechanistic insight. Dietary interventions, including caloric restriction, intermittent fasting or time-restricted feeding, have shown promising improvements in patients’ overall metabolic profiles , and an increasing number of studies have associated these beneficial effects with epigenetic alterations. In this article, we review epigenetic changes involved in both metabolic diseases and dietary interventions in primary metabolic tissues  in hopes of elucidating potential biomarkers and therapeutic targets for disease prevention and treatment. The continuous rise in metabolic diseases, such as obesity, type 2 diabetes T2D;  and non-Overnutrition, especially of highly processed foods , accompaThese dietary interventions involve limiting food intake of entire  or selected nutrient compositions , without disturbing energy balance or inducing malnutrition. Specifically, fasting interventions can be categorized into intermittent fasting (IF) and periodic fasting (PF), where food intake is limited either on a daily/weekly basis or on a monthly basis, respectively . IF cyclad libitum   .These benefits conferred by dietary interventions involve cellular adaptations within various metabolic tissues, which are mediated by epigenetic modifications. Due to the plasticity of epigenetic factors, environmental changes, such as dietary interventions, which alter food intake and composition, have a significant impact on the epigenome. In this article, we will first review epigenetic changes in metabolic disease with a particular emphasis on adipose tissues, liver, and pancreas. We will primarily focus on DNA methylation and post-translational histone modifications , with thObesity is a serious metabolic disease that has reached worldwide rates of over 27.5% for adults and 47.1% for children . It has Uncontrolled adipose tissue expansion and accompanying dysfunction drive obesity and associated metabolic pathogenesis . AdipocyAdipose tissue is classified into white adipose tissue (WAT) and brown adipose tissue (BAT). WAT stores excess energy in the form of TG and is localized to subcutaneous  and visceral depots . BAT, by contrast, utilizes stored energy to produce heat in response to stimuli like cold stress, primarily via the uncoupling protein-1 (UCP-1), in a process known as non-shivering thermogenesis . BAT is Under feeding conditions, adipocytes take up and store circulating glucose and fatty acids (FA) through processes mediated by insulin-stimulated glucose transporter type 4 (GLUT4) translocation to the adipocyte cell surface and lipoprotein lipase (LPL) activity, respectively. Under fasting conditions, adipocyte lipolysis leads to FA release into circulation for use in other metabolic tissues, including muscle, kidney, gut and liver . Adiposeob/ob) mice revealed that methylation changes are more abundant in visceral than subcutaneous adipocytes  mice compared to chow-fed and wild-type lean mice  mice  catalyze DNA methylation. In contrast, ten-eleven translocation (TET) proteins catalyze the initial step of the reverse reaction . Alterede humans . Similarean mice . Dnmt1 end IL-1β . A knownponectin . Over-ex reverse , suggestob) mice  and the diposity . Using a and BAT . Howevermeasured .Histone methylation, mediated by histone methyltransferases (HMTs) and reversed by histone demethylases (HDMs) , regulatLPL, SREBF2, SCD1, PPARG) (E2F1), a contributor of obesity pathogenesis  in association with the pax transactivation domain interacting protein (PTIP) co-factor  and LSD1 in adipose tissues or BAT specifically  exhibit similar obesogenic phenotypes with increased body weight and fat mass as well as dysfunctional BAT, indicated by lipid accumulation and reduced mitochondrial fatty acid oxidation (FAO) , a gene activation mark, is found to be enriched at the promoters of genes associated with adipogenesis and lipid metabolism  , as wellogenesis . This fio-factor . Additioo-factor . LSD1 ale tissue . Increase tissue . Notablyon (FAO) . AltogetG9a  and Ehmt1  knockout mice develop increased adiposity  and stimulate thermogenic function. Consequently, abrogation of Lsd1  (Jhdm2a (Jhdm2a–/–)  and EHMT1 are histone H3 lysine 9 (H3K9) di- and tri-methyltransferases involved in maintaining the H3K9me2/me3 repressive mark. G9a inhibits adipogenesis while promoting the activation of Wnt/β-catenin signaling  involveddiposity , while asistance . Intereselopment . In contelopment , and Jhdelopment , 2018 caox/flox)  or Jhdm2dm2a–/–)  in mice Wnt1, −6, −10a, −10b) to inhibit Wnt/β-catenin signaling while simultaneously promoting adipogenesis through the upregulation of Pparg and Cebpa (Trimethylation of histone H3 at lysine 27 (H3K27me3), a repressive mark, is increased in the WAT of HFD-fed mice and obese patients . H3K27mend Cebpa . GSK126-nd Cebpa .Dimethylation of histone H3 at lysine 36 (H3K36me2), an activation mark, is found to be protective against impaired adipose tissue function associated with obesity. The nuclear receptor binding SET domain protein 2 (NSD2) HMT mediates dimethylation at H3K36 for the activation of PPARγ-dependent gene programs, critical for mature brown and white adipocyte function. The depletion of the Nsd2-mediated H3K36me2 mark in adipocytes disrupts thermogenic function with “whitening” of BAT and increases insulin resistance of WAT .Atf3), associated with insulin resistance  and histone deacetylases (HDACs), respectively . Total Hsistance . Additioactivity , suggestpatients , however+-dependent class III HDAC that catalyzes the removal of acetyl groups from protein substrates. SIRT1 is often termed the “master metabolic regulator” due to its ability to modulate the expression of several key metabolic transcription factors and co-factors in response to environmental stimuli  (Sirt1 heterozygous mice (+/–Sirt1) , and obese patients with decreased adipose SIRT1 expression (Sirt1 deficiency is associated with increased H3K9 acetylation of TNFα and IL-1β promoter sites (Sirt1 heterozygous mice (+/–Sirt1) present with more severe insulin resistance, compared with wild-type mice , the mammalian ortholog of the yeast silent information regulator 2 (Sir2) protein, is a nuclear NAD stimuli . SIRT1 g stimuli , 2010 as stimuli . SIRT1 k SLC2A4) . Moreoveirt1+/–) , adiposepression  all exhipression  in WAT. er sites  and reduer sites . In addiype mice , which mype mice . These H+-dependent class III HDAC, which in contrast to the nuclear SIRT1, is primarily cytoplasmic, but can transiently shuttle to the nucleus for deacetylation of transcription factors , cholesterol homeostasis , blood glucose     and glucose  homeostasis and epigenetic regulation  (ENC1), previously associated with obesity  and food intake show reductions in global DNA methylation levels and differentially methylated genes associated with obesity and T2D in adipose tissues , thus pr1, INSR) , adipose, RPTOR) , and epi DNMT3B) . Studies, PRDM8) . In part obesity , was bot obesity . In anotn levels . Additioipocytes . AltogetUcp1, Pgc-1α, Ppara, Cidea) in WAT for the development of thermogenically active beige adipocytes. Consequently, whole-body Lsd1 over-expressing mice (Rosa26-Lsd1) present with reduced body weight gain and increased energy expenditure, associated with smaller adipocyte size and greater mitochondrial content in WAT    at the e levels . Additioe levels . The HATAngptl4) , 2014. IAngptl4) , HDAC3 (Angptl4) , HDAC9 (Angptl4) ,b and HDAngptl4)  negativeAngptl4)  and geneAngptl4) ,b and HDAngptl4)  in HFD-fAngptl4) , improveAngptl4) , and incAngptl4) . These m, Cidea) . Therefo, Cidea)  and hypo, Cidea) , it woulPparg promoter to co-repress target genes involved in TG storage    and redu, Mcp-1) . AltogetMcad, Lcad, Cpt1b, Cpt1a)  upon cold exposure, in comparison to wild-type mice. The post-translational SIRT1-dependent lysine deacetylation of PPARγ and its interaction with the browning co-factor Prdm16 allow for this thermogenic remodelling of WAT  . Additiog of WAT . These sg of WAT , howeverMnSOD), the apoptotic clearance of damaged cells (Bim) and the inhibition of cell proliferation and propagation of mutations (p27kip1)  .de novo lipogenesis (DNL) pathways remain insulin sensitive, contributing to hyperglycemia and hepatic lipid accumulation, respectively , DNL , and insulin signaling (PRKCE) in obese and T2D patients , appears to be central to hepatic disease progression, as PDGF-AA causes insulin resistance by reducing hepatic insulin receptor density in a protein kinase C (PKC)-dependent manner  involved in liver fibrosis  are often found in mice and patients with NAFLD and NASH , leading to ultrastructural defects in mitochondrial morphology . In addiand NASH . Consistelopment . Togethein vitro (HepG2 cells containing FA medium) and in vivo (HFD-fed mice) models of NAFLD (Tet1 (Tet–/–) in mice further exacerbates HFD-induced NAFLD, indicated by increased intrahepatic TG levels. This study suggests that hepatic Tet1-mediated hydroxymethylation of the PPARα promoter enhances FAO and thereby prevents NAFLD development.In addition to changes in genomic DNA methylation, differential expression of DNA methylation regulators has been associated with the development of hepatic diseases, particularly the TET proteins involved in hydroxymethylation, the initial step in the reversal of DNA methylation. TET1 expression is reduced in both of NAFLD . Loss ofTGFB1, to promote profibrotic gene expression and subsequent ECM production. In contrast, siRNA-mediated TET3 knockdown ameliorates liver fibrosis in mice, suggesting its crucial role in the pathological development and progression of hepatic fibrosis. This transdifferentiation of HSCs, largely regulated by a number of epigenetic processes including DNA methylation as described here, and post-translational modification of histones, is reviewed in detail elsewhere , a H3K9 HMT, is markedly reduced in genetically induced  and HFD-fed obese mice  by demethylating the H3K9me2/me3 transcriptional repressor marks and leading to increased chromatin accessibility , involved in establishing the H3K9me activation mark, is upregulated in livers of diet-induced obese mice, resulting in increased hepatic PPARγ2 expression and induction of hepatic steatosis  expression and accumulation of monounsaturated fatty acids. Conversely, Phf2 silencing leads to liver fibrosis upon a high-fat, high-sucrose diet. With supporting human data, this study suggests Phf2 as a targetable epigenetic checkpoint to prevent NAFLD progression   for transcriptional repression, also plays a key role in liver diseases. EZH2 expression is reduced in the liver of NAFLD rats and FA-treated HepG2 hepatocytes and is inversely correlated with lipid accumulation and inflammatory marker expression . The ste Col1a1) .Cd36, Fabp4, Mag, Plin2, and Fsp27/Cidec, and thereby promoting steatosis  and glycolytic  genes , leading to hypoacetylation of H3K9ac and H3K36ac  genes . The act H3K36ac . TogetheHdac3 depletion, mice develop hepatosteatosis with increased DNL, suggesting a critical role of histone acetylation-mediated circadian changes in the prevention of NAFLD. Additionally, HDAC8 has been identified as a commonly upregulated gene in dietary and genetic obesity-promoted HCC mouse models as well as in human HCC cells and tissues , lipid synthesis  and lipid elongation . These changes increase insulin sensitivity while reducing lipid content and chain length of TG-associated FA in the livers of old female mice (Irs2) promoter, thereby repressing Irs2 expression. CR has been shown to improve insulin resistance in mice through hypermethylation and downregulation of Srebp1 as well as through hypomethylation and upregulation of Irs2, the direct target of SREBP1    in the mouse liver results in reduced hepatic β-oxidation and increased steatosis. These data suggest a critical role for histone methylation modulators in mediating the metabolic improvements associated with fasting-related dietary interventions against liver metabolic dysfunction.Dietary interventions, such as fasting, can modulate hepatic histone methylation and subsequent gene transcription. In particular, fasting-induced protein expression of JMJD3 (KDM6B), a H3K27me3 HDM , stimulaa, Mcad)  and autol, Atgl)  in the ll, Atgl)  or PKA pl, Atgl) . The fasHepatic histone acetylation in the context of dietary interventions mainly involve HDACs. Specifically, under fasting conditions, class IIa HDACs upregulate the hepatic gluconeogenic gene program. Fasting-induced glucagon-secretion in primary mouse hepatocytes promotes protein kinase A (PKA)-mediated phosphorylation and inactivation of SIK2, thereby allowing for the nuclear translocation of unphosphorylated class IIa HDACs . Nuclear+/NADH ratio activates hepatic SIRT1, a class III HDAC. The genetic over-expression of SIRT1 or its activation by resveratrol treatment protects against HFD-induced hepatosteatosis and glucose intolerance  and β-oxidation  genes, while repressing glycolytic genes  (Sirt1 results in the hyperacetylation of PGC-1α at PPAR response element (PPRE) sites on target genes, thereby inhibiting PPARα signaling , an increase in the NADolerance  as well olerance . Mechanipk, Gck) . SIRT1-dpk, Gck) . Hepatocignaling . Additioignaling . Inhibitignaling . SIRT1 aignaling , for theSirt1 over-expression in mice concluded that despite functional similarities such as improved insulin sensitivity, Sirt1 gain-of-function does not mimic nor boost the metabolic effects of IF , but not short-term fasting (6−8 h), and thus may play a role in the later stages of nutrient depletion  and ts of IF . This suts of IF , some stts of IF  or no chts of IF  in expreH levels . Furthereostasis  – an effeostasis  upon Sirepletion . Some diepletion  and pancepletion  occur duThe pancreas is a secretory organ with both exocrine (acinar) and endocrine (islet) function. Among the pancreatic islet cell types, the glucagon-secreting α-cells and the insulin-secreting β-cells are primarily involved in regulating the metabolic pathways of the fed and fasted states. Under fasting conditions, glucagon is released to increase blood glucose levels through the promotion of hepatic gluconeogenesis and glycogenolysis, whereas under feeding conditions, insulin is secreted to promote the uptake of glucose, amino acids and FA and to stimulate processes of glycogenesis, protein synthesis and DNL in insulin-sensitive metabolic tissues . In the INS) at 4 CpG sites, correlates negatively with insulin mRNA levels and positively with glycated hemoglobin HbA(1c) levels, which reflect the cumulative blood glucose concentration (in vitro model of a rat β-cell line (INS 832/13), 72-h of high glucose exposure (16.7 mmol/L), which recapitulates conditions of hyperglycemia in T2D, increases DNA methylation within the Ins promoter  , a master transcriptional regulator of β-cell development and function, is reduced with increased methylation in pancreatic islets of T2D patients, which also corresponds to increased HbA1c levels (GLP1R), involved in enhanced GSIS upon GLP-1 peptide binding  . A 48-h T2D risk . These mc levels . Similar binding , is decr binding . In partnd HbA1c , suggestecretion .loci associated with the T2D risk and accounting for ∼10% of heritable diabetes  in human islets. Together, these studies demonstrate that DNA methylation may provide a causal link between SNPs and pancreatic gene function, thereby contributing to T2D development and progression , which mildly reduces HDAC activity, has been found in type 1 diabetes (T1D) patients and leads to hyperinflammation with elevated expression of nitric oxide, cytokines , and chemokines in a β-cell line (MIN6)  present with glucose intolerance and impaired GSIS of β-cells  . Pancrea β-cells . SIRT1-mtes GSIS , howeverIS  improves glucose tolerance and insulin sensitivity  (ob/ob) mice improves glucose homeostasis with increased postprandial insulin secretion, particularly GSIS (db/db) mice confers improvements in β-cell function as indicated by decreased plasma glucose and increased plasma insulin levels, as well as a reduction in insulin resistance  in rodent models of diabetes  . In partrly GSIS . As IF isistance . Notablyts on CR .Lsd1 results in histone hyperacetylation accompanied by insulin hypersecretion, indicating that the adaptive insulin secretory response to dietary interventions is regulated by the modulation of the epigenome in the pancreas  in mice enhances GSIS in isolated islets without affecting body weight  and is apancreas .Sirt1 over-expression in mice preserves glucose homeostasis through improvements in insulin secretion and glucose tolerance (Ucp2 increases cellular ATP to promote vesicular exocytosis and release of insulin from β-cells (SIRT1 over-expression or resveratrol (CR-mimetic) treatment post-translationally deacetylate both p65 for inhibition of the NF-kB inflammatory signaling pathway  and the function of these microorganisms make up the microbiome . While tGut microbiota influences the host metabolism through various microbial-derived metabolites, which induce epigenetic alterations of key genes involved in the initiation and progression of diseases . MetabolLactobacillus bacteria population, resulting in increased levels of serum lactate (Mct1) in WAT  in WAT . Lactobadiseases  and is iribacter . Notablyribacter , suggestribacter , the assMany of the benefits related to fasting and nutritional interventions, such as IF, CR, FMD and KD, may stem from the activation of the ketogenic pathway and the increased production of ketone bodies . This metabolic switch to ketosis, associated with improvements in lipid and glucose metabolism, contributes to a healthier metabolic state of the tissues discussed in this review. Dysregulation or insufficiency of ketogenesis, by contrast, can be associated with hepatic metabolic abnormalities and may contribute to NAFLD  and liveHMGCS2 is a downstream target gene of FOXA2, a key transcription factor of hepatic lipid metabolism. Under feeding conditions, insulin/PI3K/Akt-mediated phosphorylation of FOXA2 reduces its transcriptional activity by nuclear exclusion  is the rate-limiting enzyme of the ketogenic pathway and catalyzes the conversion of acetoacetyl-CoA to HMG-CoA – the first step in β-hydroxybutyrate (BHB) synthesis. xclusion . Converscription . In addicription . Mice laovements .Hdac3-deficient mice , stress response, and longevity induction  and BHB are the three ketone bodies produced by the liver, from which AcAc and BHB are transported primarily to skeletal muscle and the brain as carriers of additional energy, while acetone is mainly released through exhalation. Among the three ketone bodies, BHB is the most abundant in mammals. Notably, recent studies have demonstrated that ketone bodies play a pivotal role as direct or indirect signaling mediators of cellular and metabolic functions, including epigenetic gene regulation . Similarent mice . In addinduction . Since bnduction , 2018 annduction  in both Furthermore, elevated BHB levels result in increased histone lysine β-hydroxybutyrylation (kbhb), a novel type of histone post-translational modification . BHB proGpr43), activated specifically by AcAc in mice, abolishes IF- and KD-mediated metabolic benefits, including those associated with lipid metabolism   inhibitors, are being clinically used in the treatment of NAFLD and T2D . SGLT2 iduction) . This fiIn this review, we summarized key tissue-specific epigenetic changes implicated in both metabolic diseases and dietary interventions, with a focus on adipose, liver and pancreas. While most of the epigenetic studies have been conducted separately in these metabolic tissues, it is important to recognize that the integrated tissue cross-talks can drive systemic changes in metabolic gene expression and function. For example, secretion of tissue-respective metabolites and hormones, such as adipokines , hepatokines  and pancreatic glucagon and insulin, which as we have discussed are all subject to epigenetic changes, are key mediators of tissue cross-talk and systemic homeostasis . TherefoOne of the key limitations of dietary interventions arises from sex differences. Despite the successful outcomes of dietary interventions seen in humans and animal models , it is nAging is a key risk factor for metabolic disease development. Extension of lifespan and healthspan by dietary interventions has thus led to an interest in their application for the treatment of metabolic diseases in the elderly . At the in vitro and in vivo mechanistic studies of current epigenetic therapies, such as inhibitors of DNMT   , 2010, Hytidine)  and HAT ytidine)  have shoytidine) . Interesytidine) . Resveraytidine) . AlthougSA, NM, EM, and K-HK conceived and wrote the manuscript. SA and NM created the figures and table. All authors read and approved the final manuscript.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.


Chromosome instability (CIN), the hallmarks of cancer, reflects ongoing chromosomal changes caused by chromosome segregation errors and results in whole chromosomal or segmental aneuploidy. In multiple myeloma (MM), CIN contributes to the acquisition of tumor heterogeneity, and thereby, to disease progression, drug resistance, and eventual treatment failure; however, the underlying mechanism of CIN in MM remains unclear. Faithful chromosomal segregation is tightly regulated by a series of mitotic checkpoint proteins, such as budding uninhibited by benzimidazoles 1 (BUB1). In this study, we found that BUB1 was overexpressed in patient-derived myeloma cells, and BUB1 expression was significantly higher in patients in an advanced stage compared to those in an early stage. This suggested the involvement of aberrant BUB1 overexpression in disease progression. In human myeloma-derived cell lines (HMCLs), BUB1 knockdown reduced the frequency of chromosome segregation errors in mitotic cells. In line with this, partial knockdown of BUB1 showed reduced variations in chromosome number compared to parent cells in HMCLs. Finally, BUB1 overexpression was found to promote the clonogenic potency of HMCLs. Collectively, these results suggested that enhanced BUB1 expression caused an increase in mitotic segregation errors and the resultant emergence of subclones with altered chromosome numbers and, thus, was involved in CIN in MM. Multiple myeloma (MM) is a cytogenetically and molecularly heterogeneous hematological malignancy that originates from plasma cells and remains mostly incurable, despite recent therapeutic advances. MM is invariably preceded by the precursor state of monoclonal gammopathy of undetermined significance (MGUS), which progresses to smoldering MM and later to MM. Therefore, MM provides an excellent model through which understanding of the multistep evolutional process of cancer progression might be achieved. Early events include acquisition of hyperdiploidy commonly characterized by trisomy of certain odd-numbered chromosomes 3, 5, 7, 9, 11, 15, 19, 21, or immunoglobulin heavy (IGH) chain gene chromosomal translocations. Subsequent to the primary event, numerous types of secondary cytogenetic or molecular aberrations, including chromosomal translocations, copy number abnormalities, somatic mutations, and epigenetic alterations, emerge in an overlapping manner in myeloma cells. This multistep acquisition of a series of genetic abnormalities gives rise to subclones with a selective advantage for disease progression, even under therapeutic pressure ,5,6. TheFaithful segregation of replicated chromosomes during mitosis is essential for the maintenance of genomic integrity and, therefore, is tightly regulated by the mitotic checkpoint  mechanism, which requires the coordinated functions of a series of mitotic checkpoint proteins, such as budding uninhibited by benzimidazoles 1 (BUB1), BUB3, BUBR1, mitotic arrest deficient 1 (MAD1), MAD2, and monopolar spindle 1 (MPS1) ,11. ThesThe serine/threonine kinase BUB1 is a highly conserved multifunctional protein that is essential for the mitotic spindle checkpoint and correct kinetochore-microtubule attachments . In addin = 3), patients with MGUS (n = 19), newly diagnosed MM (NDMM) (n = 12), and relapsed/refractory MM (RRMM) (n = 27) was examined to investigate its relationship with disease status. BUB1 mRNA tended to be higher in CD138-positive myeloma cells from MGUS and NDMM compared to normal plasma cells, but the difference was not significant. However, the BUB1 mRNA levels in myeloma cells from patients in an advanced stage RRMM and HMCLs were significantly higher than that in normal plasma cells and higher than that in myeloma cells from patients with early-stage disease (MGUS and NDMM). The BUB1 mRNA level did not differ significantly between MGUS and NDMM , and the second-lowest BUB1 protein among seven HMCLs examined, respectively. We excluded KMS-12-BM with the highest and NCI-H929 with the lowest BUB1 protein to avoid extreme situations a,c. BUB1The oncogenic function of BUB1 in cell proliferation is unclear among cancer types ,29,30,31We next investigated the functional involvement of BUB1 overexpression in the acquisition of chromosomal abnormality in MM. For this purpose, we examined the impact of BUB1 knockdown on anaphase chromosome segregation errors in KMS-18 and AMO-1 cells that possess highly complex chromosomal abnormalities, including both numerical and structural abnormalities. In addition, chromosomal features were highly variable among individual metaphase spreads in both HMCLs. In brief, most of the KMS-18 cells harbored more than 60 chromosomes and showed various abnormalities, including chromosome (chr) 1q gain, chr13 deletion, t for IGH/MMSET, and t for IGH/MYC, while most of the AMO-1 cells possessed more than 80 chromosomes and abnormalities, such as chr1q gain and t . Thus, tChromosome segregation errors were classified into two categories: attachment defects, such as lagging chromosomes with centromeres, and structural defects, including acentric chromosomes and chromosome bridges a. ApproxGiven that the BUB1 expression level determined the frequency of anaphase chromosome segregation errors, we next examined whether the reduction of these errors by BUB1 partial knockdown resulted in a change in the number of chromosomes in a single cell. For this purpose, KMS-18 and AMO-1 cells were subjected to limiting dilution in order to select 10 single-cell clones, which were cultured until the single-cell expanded to 5 million cells (a division of 20 times or more). The numbers of chr3 and chr7 were then counted as representatives of all chromosomes by FISH analysis ,34 in 10To evaluate the impact of BUB1 knockdown on total chromosome numbers in more detail, we performed G-banding to count total chromosome numbers, including supernumerary chromosomes and fragments, in 50 mitotic cells of parental and BUB1-reduced KMS-18 and AMO-1 cells. The average total chromosome numbers were not significantly altered by BUB1 knockdown, but standard deviations were significantly reduced by BUB1 reduction in both HMCLs b. CollecIn breast cancer cells, the BUB1 knockdown does not affect short-term cell growth but does impair clonogenic potency . This prMM remains a hard-to-treat disease despite the advent of new therapeutic strategies in the past two decades. One underlying reason for the difficulty in the development of a curative drug for MM is the lack of a universally targetable molecule due to the high interpatient diversity and the intraclonal heterogeneity of cytogenetic and molecular abnormalities in a single patient ,4,5,6,7.More importantly, additional cytogenetic abnormalities due to CIN occur in subclones of myeloma cells. As in many other cancers ,38,39,40Aberrant expression of BUB1 has been repeatedly reported in various cancers ,43,44,45The disease aggressiveness-associated BUB1 overexpression may be translated into the development of a new biomarker for MM. It would be interesting to investigate if the BUB1 expression level may be a surrogate for the degree of CIN in myeloma cells, and thereby, constitute a new prognostic marker. In addition, while we investigated BUB1 expression in CD138-positive patient-derived myeloma cells in this study, it would be also interesting to investigate BUB1 expression in CD138-negative myeloma cells, considering the occasional loss of CD138 expression in myeloma cells of the more advanced clinical phase. However, while AURKB and receptor for hyaluronan-mediated motility (RHAMM) have been suggested to interact with BUB1 to promote cancer stemness and tumor formation ,44, we wThere are some limitations to the study. We showed the involvement of BUB1 in CIN in short-term observation in HMCLs. However, parental KMS-18, AMO-1, and RPMI-8226 cells did not show a change in total chromosome numbers of the modal clone after several weeks with cell division of over 20 times b and notIn conclusion, this study revealed the possible association of BUB1 overexpression with CIN in MM. A further study is needed to determine the mechanism linking the clonal evolution of myeloma cells with BUB1 dysregulation, which may lead to new diagnostic and therapeutic approaches to limit myeloma progression.n = 19), NDMM (n = 12), and RRMM (n = 27) between March 2014 and March 2018 at Kyoto Prefectural University of Medicine and Gunma University. MGUS/MM was diagnosed based on the International Myeloma Working Group 2014 criteria [Bone marrow samples were obtained from patients with MGUS (criteria . Bone maRRMM n =  between 2 in the air.Ten human myeloma-derived cell lines (HMCLs) were used in the study. AMO-1, NCI-H929, OPM2, and LP-1 were obtained from Deutsche Sammlung von Mikroorganisem und Zellkulturen , and RPMI-8226 and IM9 were from American Type Culture Collection . KMS-18, KMS-12-BM, KMS-28-PE, and KMS-34 are gifts from Dr. T. Ohtsuki . Most HMCLs were derived from an aggressive form of MM patients, such as with extramedullary disease or leukemic transformation . Cells wTotal RNA extraction and quantitative RT-PCR (qRT-PCR) were performed, as described elsewhere ,32,52,53Western blotting was performed, as described previously ,32,52,53BUB1 silencing was accomplished by stable expression of inhibitory short hairpin BUB1 RNAs (shRNAs). The target sequences for human BUB1 were 5′-GCA ACA ACA ATA CAG GTT ATT-3′ (shBUB1#1) and 5′-GCC TGA AGA TAC ATT CTA TTT-3′ (shBUB1#2). The sequences encoding shRNAs against BUB1 were cloned into lentivirus vector pLKO.1 puro . The empty vector was used as negative control (mock). Lentivirus was produced by transient co-transfection of HEK293T cells with a lentivirus vector, packaging plasmid pMDLg/pRRE, pRSV-Rev, and pMD2.G using PEI MAX . Viral supernatant was collected 48 and 72 h after transfection and concentrated by ultracentrifugation. Infected cells were selected with 5 μg/mL puromycin beginning 48 h after infection and maintained under selection for 3–4 passages.Cells were spun onto glass slides in a cytocentrifuge using Cytofuge 2  for 6 min at 1000 rpm, fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton X-100, and blocked with 5% BSA in PBS for 20 min. Cells were incubated with anti-CENP-C antibody  overnight at 4 °C and then incubated with goat anti-Guinea Pig IgG (H + L) secondary antibody, Alexa Fluor 546 , and 4′,6-diamidino-2-phenylindole (DAPI) for 1 h. Glass slides were then rinsed and mounted with ProLong Gold antifade reagent (Invitrogen) for imaging. A total of 100 cells were inspected using a fluorescence microscope BZ-X710 .Conventional G-banding for metaphase spreads and fluorescence in situ hybridization (FISH) studies were performed, as described elsewhere . Interph5 cells/mL in a 24 well-plate, and cell number was counted by Trypan blue dye exclusion. For cell cycle analysis, cells were permeabilized and fixed with ice-cold 70% ethanol overnight, stained with propidium iodide (Sigma), and then subjected to flow cytometric analysis using a FACS Calibur . Data obtained were analyzed using FLOWJO software Ver. X  [For the evaluation of cell proliferation, cells were seeded at 1 × 10, Japan) .2 in the air, numbers of colonies were counted on an inverted microscope, and then colonies were fixed with methanol and stained with crystal violet dye.KMS-18 cells were plated in Methocult H4230  with 6% fetal bovine serum and 0.2% bovine serum albumin at 2000 cells/mL in triplicate dishes. RPMI-8226 cells were plated in the same medium at 250 cells/mL in triplicate dishes. After incubation for 14 days at 37 °C in a fully humidified atmosphere of 5% COt-test. The distributions of whole chromosome numbers of 50 metaphase cells among HMLCs were compared by the F test. p < 0.05 was considered significant in all analyses.Data are expressed as mean ± SD of independent triplicate experiments. The significance of differences in the expression level of BUB1 was determined by one-way ANOVA with a Tukey multiple comparison test. Segregation errors, chromosomal number, cellular growth, and colony-forming ability were evaluated by the Student In this study, we identified aberrant overexpression of BUB1 in myeloma cells, which associates with disease progression and aggressiveness. Our study suggested that enhanced BUB1 expression caused an increase in mitotic segregation errors and the resultant emergence of subclones with altered chromosome numbers, and thus, was involved in CIN in MM. Understanding the functional significance of BUB1 overexpression may lead to new diagnostic and therapeutic approaches to overcome myeloma progression.


Trichomonas vaginalis requires the availability of targets with high specificity. Such targets should comprise epitopes unique to T. vaginalis immunogenic proteins detected by sera of women and men patients with trichomonosis but not uninfected controls. Three enzymes to which patients make serum IgG antibody were identified as fructose-1,6-bisphosphate aldolase (A), α-enolase (E), and glyceraldehyde-3-phosphate dehydrogenase (G). Epitopes within these proteins were identified that had no sequence identity to enzymes of humans and other pathogens. Therefore, I constructed a chimeric recombinant String-Of-Epitopes (SOE) protein consisting of 15-mer peptides, within which are the epitopes of A, E, and G. This novel protein of ~36-kD is comprised of two epitopes of A, ten epitopes of E, and seven epitopes of G (AEG::SOE2). The AEG::SOE2 protein was detected both by immunoblot and by enzyme-linked immunosorbent assay (ELISA) using highly reactive sera of women and men but not negative serum unreactive to T. vaginalis proteins. Finally, AEG::SOE2 was found to be immunogenic, as evidenced by serum IgG from immunized mice. I discuss how this approach is important in relation to infectious disease diagnostic targets for detection of serum IgG antibody in exposed and/or infected individuals and how such novel targets may have potential as subunit vaccine candidates against microbial pathogens.Point-of-Care (POC) serum antibody screening of large cohorts of women and men at risk for the sexually transmitted infection (STI) caused by  Trichomonas vaginalis causes a non-viral sexually transmitted infection (STI) with adverse outcomes to infected women [T. vaginalis DNA have been detected in hyperplastic prostate tissue [T. vaginalis in relation to prostate cancer (PCa) development [T. vaginalis-mediated PCa was proposed [ed women ,2. This ed women ,4,5, ande tissue ,7, and telopment ,9,10. Moproposed , and othproposed ,13,14,15TM Trichomonas Rapid Test, Sekisui Diagnostics, Lexington, MA, USA) for rapid detection of active trichomonosis in women was invented in my laboratory [A rapid, inexpensive and specific serodiagnostic that could be used for screening large cohorts of at-risk individuals is desirable. A lateral flow, immunochromatographic Point-of-Care (POC) diagnostic  can be synthesized as a serodiagnostic target. My laboratory has previously determined that women and men patients make serum IgG antibody to numerous T. vaginalis immunogenic proteins, including the enzymes fructose-1,6-bisphosphate aldolase (referred to as A), α-enolase (E), and glyceraldehyde-3-phosphate dehydrogenase (G) [As infection by response ,10,11,21nase (G) ,23. Epitnase (G) ,10,24,25® accession numbers AAW78351 (A), AAK73099 (E), and AAA30325 (G). The protocols for probing of SPOTs membranes with sera were as detailed before [www.iedb.org) to show the linear nature of the peptide–epitope sequence.The identification of immunogenic epitopes reactive to women and men patient sera was done using oligopeptides (Custom Peptide Arrays) immobilized on membranes  as recently detailed ,24. As bd before . The epid before . Furtherd before . FinallyT. vaginalis [The α-actinin protein is one of the most immunogenic proteins of aginalis ,27 and haginalis . The truaginalis . ACT-P2 aginalis  and moreaginalis ,9,10. Thaginalis .+) and chloramphenicol (Cam +) genes. The recombinant E. coli BL21DE3 cells were used to synthesize the protein. The description of ACT-P2 in E. coli has been described [The DNA coding sequence for the chimeric, recombinant AEG::SOE2 protein was synthesized by GenWay Biotech, Inc . The pET-23a (+) plasmid that was prepared was encoded for ampicillin , and soluble AEG::SOE2 was recovered from E. coli lysates using the Ni2+-NTA column, as evidenced by Detailed protocols for growing bacteria on Luria Broth (LB) agar plates with either 25 µg/mL Kanamycin (ACT-P2) or 100 µg/mL Ampicillin (AEG::SOE2) have been published [s before ,22,23,24s before ,24,25. PNumerous reports have described the sera from women and men used for detection of ACTP2 ,24. SeraMy laboratory has published detailed descriptions for the use of sera by ELISA for detection of proteins, and identical materials and methods published earlier were used in this study to compare reactivities with ACTP2 and AEG::SOE2 ,10,21,25In order to perform comparative analyses of ELISA using different targets, it was necessary to have serum standards as negative  and positive (scored as 3+ and 4+) controls derived from testing ACTP2 as the gold standard for screening ,10,24,25T. vaginalis serum was obtained by immunizing BALB/c mice as previously described [Mouse anti-escribed . Mouse a5 organisms was added to individual wells of microtiter plates. After drying at 37 °C, 50 µL of 95% ethanol was added as fixative and wells were allowed to dry. Both protein-and trichomonad-coated wells were washed three times with PBS-Tween followed by blocking with 200 µL of eBSA-PBS. The remaining standard protocol is as previously reported elsewhere [We also performed a whole cell ELISA for detecting antibody using microtiter wells coated with trichomonads. Parasites at logarithmic growth were washed three times with PBS, and a 50 µL suspension containing 1.25 × 10lsewhere ,24,25.p-value less than 0.05.All experiments were performed at least three times under identical conditions. ELISAs on microtiter plates coated with proteins or trichomonads were done in quadruplicate unless otherwise indicated, and means and standard deviations were calculated. All statistical analyses were conducted with RStudio , and figures were made in Prism . T-tests were used to examine differences in absorbance levels for ACTP2 and AEG::SOE2 for mouse sera, negative and positive human sera, and monoclonal antibodies. Statistical significance was defined as a T. vaginalis A, E, and G proteins, respectively, that had no sequence identity with other bacterial, fungal, parasite, and human sequences [T. vaginalis proteins had identity, albeit to different degrees, with protein sequences of enzymes of other bacterial, fungal, parasite, and human proteins (data not shown).Epitope mapping revealed that pooled women and men positive serum recognized a total of 12 epitopes of A, 18 epitopes of E, and 19 epitopes of G, for a total of 49 epitopes for the three proteins . Table 1equences . The redr of 35,896.31 daltons and pI of 5.05. Overall, there are 8 and 11 epitopes detected by positive women and men sera, respectively. The four monoclonal antibodies (MAbs), labeled ALD 13, ALD 55, ALD 32, and ALD 30, are reactive with the epitopes, as indicated, and these MAbs have been previously characterized [cterized .T. vaginalis serum [A) and 5 µg (B) of protein on wells were detected by the mouse antiserum at a 1:100 dilution (wells numbered 2), the cocktail of IgG1 monoclonal antibodies (MAbs) , and pooled patient sera at a 1:25 dilution (wells numbered 4). Not surprisingly, the protein  gave statistically significant higher values to both ACTP2 and AEG::SOE2 when compared with both prebleed, normal mouse sera (NMS), and secondary peroxidase-conjugated goat anti-human IgG alone (P-G anti-H IgG). Finally, and not unexpectedly, the MAbs to AEG::SOE2 (We now compared AEG::SOE2 by ELISA with the gold standard ACTP2 for detection of IgG. EG::SOE2  gave staFor clarification of what the scores signify in terms of the mean and standard deviation (SD) absorbance readings for Finally, I randomly selected 42 each of sera of women and men with different scores for side-by-side evaluation. T vaginalis serum [T. vaginalis [Finally, we wanted to examine whether mice immunized with AEG::SOE2 produced IgG antibodies. We compared mouse anti-AEG::SOE2 serum with mouse anti-is serum  in indivaginalis ,24.T. vaginalis seropositivity  [These novel, chimeric SOE proteins are comprised of immunogenic epitopes unique to the microbial pathogen of interest and, in this case, omonosis ,29,30. Taginalis ,33. I arpitopes) . In othepitopes) . Thus, tT. vaginalis acquires onto its surface numerous host serum proteins [T. vaginalis surface-associated E and G metabolic enzymes are ligands that bind host proteins, such as plasminogen, fibronectin, collagen, and laminin [T. vaginalis may have altered structures exposing epitopes to host antibody responses, creating possible auto-antibodies and adverse reactions with tissues. This possibility deserves more attention in host–parasite interactions.Another reason against whole organisms and lysates as vaccines is that we now know that proteins ,36,37,38 laminin ,23. As t laminin ,36,37,38T. vaginalis proteins. This conclusion has merit based on the findings presented here. This, then, would lead to false-positive reactions for this STI. Therefore, future serodiagnostic targets for this and other infectious diseases must have specific epitopes that are unique to the pathogen causing the disease. These concerns are relevant to the development of effective infectious disease diagnostics and vaccines and are also important considerations for surveillance and interventions of STIs and infectious diseases [Of interest is that trichomonad lysates were recently compared with α-actinin by ELISA for IgG reactivity , and botdiseases .T. vaginalis [T. vaginalis, and this may be verified as was recently shown by others for α-actinin [Finally, I have shown that the chimeric AEG::SOE2 protein is immunogenic, as evidenced by IgG antibody made by immunized mice . Importaaginalis ,23. More-actinin . I belieTrichomonas vaginalis was used as a model to test the viability of the approach. This approach includes i) identification of immunogenic surface proteins; ii) epitope mapping; iii) selection of epitopes with amino acid sequences unique to the trichomonad proteins; and iv) construction of a hybrid String-Of-Epitopes AEG::SOE2 protein comprised of A, E, and G epitopes. Finally, because of the highly immunogenic nature of the epitopes, as evidenced by patients’ serum IgG reactivities (T. vaginalis and SOE proteins of infectious agents in general may have potential vaccine applicability.A stepwise approach is presented that may have applicability for infectious diseases by producing a unique and specific serodiagnostic target to an infectious agent, and such SOE proteins can be tested for specificity as a diagnostic target. tivities , the factivities , and theJ.F. Alderete. Strings of Epitopes Useful in Diagnosing and Eliciting Immune Responses to Sexually Transmitted Infections. No. 9910042, 5 March, 2018.J.F. Alderete. Strings of Epitopes Useful in Diagnosing and Eliciting Immune Responses to Sexually Transmitted Infections. No. 10386369, 20 August, 2019.


Given the differences in embryonic origin, vascular and nervous supplies, microbiotic burden, and main physiological functions of left and right colons, tumor location is increasingly suggested to dictate tumor behavior affecting pathology, progression and prognosis. Right-sided colon cancers arise in the cecum, ascending colon, hepatic flexure and/or transverse colon, while left-sided colon cancers arise in the splenic flexure, descending, and/or sigmoid colon. In contrast to prior reports, we attempt to delineate programs of tumorigenesis independently for each side.Four hundred and eleven samples were extracted from The Cancer Genome Atlas-COAD cohort, based on a conservative sample inclusion criterion. Each side was independently analyzed with respect to their respective normal tissue, at the level of transcription, post-transcription, miRNA control and methylation in both a stage specific and stage-agnostic manner.CYP2C8, CYP4F12, GSTA1, and UGT1A within right colon tumors. This implies its reduced capacity to detoxify carcinogens, contributing to a genotoxic tumor environment, and subsequently a more aggressive phenotype. Additionally, we highlight a crucial nexus between calcium homeostasis  and immune/GPCR signaling within left-sided tumors, possibly contributing to its reduced proliferative and metastatic potential. Interestingly, two genes SLC6A4 and HOXB13 show opposing regulatory trends within right and left tumors. Post-transcriptional regulation mediated by both RNA-binding proteins (e.g. NKRF (in left) and MSI2 (in right)) and miRNAs (e.g. miR-29a (in left); miR-155, miR181-d, miR-576 and miR23a (in right)) appear to exhibit side-specificity in control of their target transcripts and is pronounced in right colon tumors. Additionally, methylation results depict location-specific differences, with increased hypomethylation in open seas within left tumors, and increased hypermethylation of CpG islands within right tumors.Our results indicate a suppression of enzymes involved in various stages of carcinogen breakdown including Differences in molecular mechanisms captured here highlight distinctions in tumorigenesis and progression between left and right colon tumors, which will serve as the basis for future studies, influencing the efficacies of existing and future diagnostic, prognostic and therapeutic interventions. KRAS, APC and/or BRAF mutations  and hypermethylation  [Colorectal cancer (CRC) is a heterogeneous disease with distinct clinical, molecular, and pathophysiological characteristics. The Surveillance, Epidemiology and End Result (SEER) program of the National Cancer Institute, in 2018, identified CRC as the fourth most prevalent cancer, and the third leading cause of mortality due to cancers, with an average five-year survival of 64.5%. Traditionally, patient subgroups in CRC are associated with dysfunction of mismatch repair genes ; e, CIMP) . To thisRight-sided colon cancers  occur in the cecum, ascending colon, hepatic flexure and/or transverse colon, while left-sided colon cancers  arise in the splenic flexure, descending colon, and/or sigmoid colon. This distinction has been observed at physiological, molecular and therapeutic levels for RSCC and LSCC . For exaTo the best of our knowledge, recent studies focusing on molecular differences between RSCC and LSCC, have done so by directly comparing the two etiologies (e.g. ). HoweveTCGA level 3 COAD raw count data for 499 solid tumor and normal tissue was downloaded via Firebrowse in July 2018. Samples were classified into right/proximal and left/distal based on their site of extraction. Since TCGA clinical data was not specifically annotated for exact location along the transverse colon, all samples annotated “transverse colon” were excluded from analysis . SamplesCount matrix normalization and differential analysis was performed using the “DEseq2” package available through R/Bioconductor . Expressp-values were obtained from univariate Cox proportional-hazards regression models for the entire list of differentially expressed genes on the right and left . All Kaplan–Meier survival curves were constructed using survival and ggplots2 packages. Median expression was utilized to separate high from low expression group in left and right samples. A total of 76 and 42 gene markers were identified to be associated with overall survival in left and right respectively . Specifically, events were defined as death by any cause, and time was accurate to the day. p < 0.05). Manually curated miRNA-target interactions from miRTarBase 7.0 [miRNA expression data was downloaded using TCGAbiolinks , across Base 7.0  between Percent splice-in (PSI) values were downloaded from TCGASpliceSeq  for our sample cohort, accounting for a total of 23,176 events. “psichomics”  availablp < 0.05) for each side independently. We utilized the entire list of DEGs on each side to identify enrichment of RBP binding using AURA2 [A combined list of 1608 RNA-binding proteins (RBPs) were extracted from supplementary tables available in two prior publications , 19, conng AURA2 . Since, β  that were at least 25% differential (|Δβ| > 0.25) between tumor and adjacent normal tissues with adj p ≤ 0.001, for each side. Correlated and anti-correlated pairs of DEGs with DMPs (Hypo and Hyper) and transcription factor (TF) enrichment were identified using ELMER [Illumina HumanMethylation450 beadchip data for TCGA-COAD, probing nearly 480 k CpG sites, was downloaded using TCGAbiolinks. Nearly 370 k probes were retained for each side, after eliminating probes on sex and “NA” chromosomes. Mean methylation and differentially methylated probes (DMPs) were both determined using the TCGAbiolinks package, independently for each side. DMPs were identified using ng ELMER .4 database [All functional enrichment analyses were performed using mSigDB’s hallmark data sets and GO ontologies. All networks were constructed, clustered, annotated and analyzed in Cytoscape ; Visualidatabase  or ELMERDifferential analysis identified 2495 DEGs in LSCC, with respect to left normal colon tissue, and 2589 DEGs in RSCC, with respect to right normal colon tissue Fig. a. 957 ub and c.AXIN2, WNT2, WNT3, WNT7B, DKK1/4, NKD1/4, TCF7, MYC and NOTUM. NOTUM, a glypican-dependent WNT inhibitor serves as a negative feedback regulator for WNT activation [NOTUM to be significantly associated with OS in patients with both RSCC (Hazard Ratio 95% CI - 0.44 (0.24–0.82), logrank p < 0.01) and LSCC (HR 95% CI – 3.23 (1.27–8.2), p < 0.01) indicating that a higher expression favors LSCC while lower expression favors RSCC .Malignant tumor cells are highly plastic and are characterized by alterations in metabolism, adhesion, proliferation and migration, requiring coordinated activity of several signaling pathways and mechanisms. Our results indicate that the large overlap of “commonly” regulated DEGs that exist between RSCC and LSCC fit broadly into these categories and are frequently seen dysregulated in colon cancers Fig. a. For intivation , and is tivation . We idenAMPK1) activity can drive reprogramming of cellular metabolism and is at the center of the network regulating cell growth and proliferation (via TP53). AMPK1 driven-metabolic dis-homeostasis becomes evident by the observed dysregulation of melanoma antigens (MAGEA2/A3/A6) [2, have been identified as crucial mediators of tumor pH [CA1/2/4) and water channels (AQP8) are suppressed in both, alluding to the reduced availability of the universal buffer HCO3−. It has been previously postulated that extracellular CAs such as CA9 (upregulated in both tumor types), act to raise the extracellular pH favoring tumor cell growth, proliferation, and survival [MUC16) and CEA  are upregulated in both RSCC and LSCC.Cellular metabolism is tightly linked with cellular growth and proliferation of tumors. Loss of AMP-activated protein kinase (2/A3/A6) , within tumor pH . Notablysurvival . SeveralOTOP2 (controlled by wild-type TP53) [OTOP3, PYY and PPIAL4; and many with limited supporting evidence, might serve as interesting candidates for future research in colon cancers  , OTOP3, GSTA1), and UDP-glucuronosyltransferase (UGT) superfamily [CYP2C8, CYP2C18 and CYP4F12, Fig. UGT1A10/A7 and A8.Liver is largely considered the major organ for biotransformation . However, there is increasing acknowledgement of extra-hepatic biotransformation  tract) and its association with GI carcinogenesis. Several families of enzymes are associated with various stages of breakdown of carcinogens within the human body including cytochrome P450 (CYP), glutathione S-transferase  and ascorbic acid transport (SLC23A1/A3) are also suppressed in RSCC. Stage specific analysis revealed a unique dysregulation of members from the CYP2C and UGT1A family within proximal tumors, specifically at early stages (T1-T2) , a pivotal rate-limiting element in the transport and uptake of glucose combined with the unique downregulation of several mitochondrial metabolic markers involved in fatty acid degradation and oxidative phosphorylation including G6PC, FABP1, CPT1A, CPT2, ACAT1, ACAA2, ACOX1, EPHX2 and EHHADH further support the more pronounced shift in metabolism away from OXPHOS, within primary proximal tumors, asserting its more aggressive state [Cancer cells preferentially use aerobic glycolysis to metabolize glucose, over mitochondrial oxidative phosphorylation (OXPHOS) characterized by increased glycolysis and lactate production. Our results suggest a more pronounced shift in metabolism in proximal tumors over distal tumors. The selective upregulation of ve state .HOXB13 and SLC6A4  within the intestine. Global loss-of-function studies for TPH1 have indicated an almost complete loss of intestinal 5-HT synthesis, implying that the observed suppression of TPH1 in both RSCC and LSCC indicates a curbed extracellular production of 5-HT [HTR3E, HTR4) and intracellular enzymes required for breakdown of 5-HT  indicates a decreased bioavailability of 5-HT in both LSCC and RSCC. In light of this, it is reasonable to observe a suppression of SLC6A4 (a ligand gated serotonin-selective reuptake transporter (SERT)), required for transport of 5-HT, such as in the case of RSCC. Though no evidence in literature exists for the differential role of SLC6A4 in proximal or distal tumors within humans, we speculate that the observed upregulation of SERT expression within distal tumors (indicative of its increased activity), suggests alternate roles for SLC6A4 and/or mechanisms controlling its expression within LSCC.Two genes appear to exhibit opposing regulation trends within RSCC and LSCC –  of 5-HT . SimilarHOXB13 is an acknowledged oncogene. Studies geared specifically towards specific tumor location, have identified suppression of HOXB13 within distal tumors [PRAC1 and PRAC2 (C17orf93), two genes genomically adjacent to HOXB13, within proximal tumors.On the other hand, l tumors  and uprel tumors , consistMS4A1 , and BACH2 , two genes previously implicated in contributing to immune landscape differences between RSCC and LSCC [CXCL13/CXCR5 (TFH cell markers) and CCL19/CCL21- CCR7 (migration and activation of immune cell types). Several of these markers including MS4A1, CXCR5, and CXCL13 are also suppressed across all stages (Stages 1–4) with respect to normal, further emphasizing their role in sustaining tumor behavior within LSCC , GNG2/4/7, EDN2/3 and ADCY2/5/9 (adenylate cyclase). Two downregulated receptors LPAR1 (Lysophoatidic acid receptor), and CASR (Ca2+ sensing receptor) involved in Ca2+ homeostasis, were also detected within this cluster [2+ signaling mediated by GPCRs, specifically chemokines, and their subsequent impact on the inflammatory signatures within distal tumors (LSCC). Notably, 8/78 genes within this cluster including LPAR1, GNG4, GNG7, PMCH, GPR18, EDNRA, GPER1 and EDN3, (hypergeometric p < 0.07), are sufficient to distinguish distal tumors, from normal distal colon as identified via recursive SVM classifier , form an important class of molecules involved in post-transcriptional regulation (PTR). We focused on utilizing two levels of -omics data for analyzing differences in PTR within LSCC and RSCC.Side-specific differential analysis of 1046 micro-RNAs (miRs) identified 325 differentially regulated miRs in RSCC and 200 miRs in LSCC, compared to their respective normal tissue see . A largeG6PC and PPARGC1. Several miRs, particularly, miR-181d and miR-576, correlate with cell cycle genes including BCL2 and CCND1. BCL2, a major regulator of mitochondrial apoptosis, has been consistently shown to be down regulated in colon (and cancer) [BCL2 expression via miR-24-2 , has been previously reported in human embryonic kidney and breast cancer cell lines [TWIST1, a primal transcription factor uniquely upregulated within proximal tumors [Increasing evidence however suggests malleable roles for miRs, with multiple targets, amplifying their inhibitory or stimulatory effects on gene regulation through positive or negative feedback loops in conjunction with other miRs. We established functionally relevant, side-specific miRNA-mRNA clusters see  in an ef cancer) . Controlll lines . Interesl tumors , whose aPER1 (negative regulator of circadian rhythm) [Only two miRs however, are uniquely regulated within distal tumors– miR-3607 and miR-29a  .MAGEA3, GFRA3, EPHA5, ANK3 and TCF7. The uniquely upregulated INHBB, which correlates with miR-34a expression in proximal tumors .Particularly interesting are clusters conserved within both RSCC and LSCC Fig. c and d. ors Fig. c, was aln = 64) occur in genes commonly dysregulated in both LSCC and RSCC, making alternative splicing a major PTR regulatory mechanism within colon cancers, including genes such as AXIN2 , and MXI1  associated with the WNT pathway, and others such as IGF2 , CXCL12 , CCL24 , and S100A2 (AP exon 3). SULT1A2  and CALD1  exhibit the highest Δmedian PSI values between healthy and tumor tissues, in both left and right. SULT1A2 (suppressed ~ 3 log2 fc in both right and left tumors) is a sulfotransferase liver enzyme involved in detoxification of a variety of endogenous and xenobiotic compounds [CALD1 is a novel target of TEA domain family member 4 involved in cell proliferation and migration , UGT1A1 (Δmedian PSI = 0.12) SRI (Δmedian PSI = − 0.64) all exhibit significant AS within right tumors.Alternative splicing (AS) is an active PTR mechanism during which mRNA is actively rearranged accounting for the observed protein repertoire of complex organisms . Utiliziompounds , while CES, exon , and MXIp < 0.05, see MSI2, MEX3A, IGF2BP1/3, ELVAL4 [RBM47, DKC1, CELF4, ELAVL3 . However, a significant anti-correlation between its expression and PSI values is observed only within distal tumors  are commonly regulated in both RSCC and LSCC with several enriched for binding among DEGs  are correlated with these specific RBPs , highlighting a possibly crucial role for them in the observed (many-to-one) regulation of transcripts within colon cancer.Additionally, we identify significant correlation between the expression of mors see . AFF2 isSEPT9, VIM, GATA4, INA, MAL, WNT  and CNRIP1 are hypermethylated in both RSCC and LSCC [Development and progression of colorectal cancer is understood to undergo several genetic and epigenetic changes. Changes in the DNA methylation is one of major epigenetic mechanisms controlling CRC . Differeand LSCC , 59, furOTOP2/3, CA1/2/4, NOTUM) is more obvious in LSCC than RSCC  from both RSCC and LSCC. We find that 33% of the downregulated genes are significantly anti-correlated with at least one hypermethylated probe and 27% with hypomethylated probes within distal tumors. On the other hand, we found a higher fraction of genes being controlled by differential methylation in proximal tumors  indicative of a role for increased hypermethylation in suppressing expression with RSCC, consistent with prior research. Interestingly however, the hypermethylation and expression states of several commonly regulated DEGs , in a side independent manner. Likewise, downregulated/hypermethylated loci are enriched for the SP1/2/3 binding motifs. TF factors including ISX (suppressed ~ 2.3 fc in both), contain these binding motifs, suggesting an observed loss of these site-specific TFs might dictate de-novo hypermethylation and suppression of its downstream targets, in a side independent manner within colon cancers.Changes in methylation state of region can be due to gain/loss of site-specific transcription factors . We empl(PRAC1) is a heavily transcribed gene of the prostate, distal colon and rectum in normal physiology [HOXB13 and PRAC2 allude to possible co-regulatory mechanisms in RSCC [Proximal and distal colon tumors are suggested to be clinically, pathologically and transcriptionally distinct within CRC , 61. Mulysiology . Studiesysiology . On the  in RSCC , emphasiLPAR1 and CASR) and immune/GPCR signaling within LSCC patients, possibly contributing to the differential immune landscape of distal tumors, and their reduced proliferative and metastatic potential.Pathways known to be altered in the pathogenesis of CRC, including WNT and MYC signaling, EMT transition, inflammatory responses , and a suppression of KRAS signaling, were identified in both RSCC and LSCC. Rather than focusing on these traditionally well-known molecular mechanisms, we investigated mechanisms that contribute differentially to the sided-ness of colon cancer. Our findings indicate unique patterns of regulation, specifically within suppressed gene sets of RSCC and LSCC. A detailed analysis suggests a reduced capacity of RSCC to detoxify carcinogens, likely contributing to a genotoxic tumor environment, resulting in a more aggressive phenotype than LSCC. Additionally, increasing evidence of altered metabolism, a critical hallmark of colon cancer, is more evident in RSCC, further lending credence to the link between metabolism and cancer aggressiveness. A larger proportion of differentially regulated miRNAs on the right further support the observation that proximal tumors are more transcriptionally active . Our resKRT7, KRT17 and KRT20) are often used as metastasis markers. Notably, we identify additional markers which exhibit preferential expression within distal and proximal tumors and may serve as putative markers for early vs late stage detection. For example, KRT31 (in early stages) and KRT40 (in late stages) is expressed in distal tumors alone, while KRT13 and KRT15 are expressed in early stages of proximal tumors. We speculate KRT5, to be a stage-agnostic marker for RSCC. Likewise, FDA approved serum tumor marker CEA, is encoded by the CEACAM family of genes. Though, CEACAM1 and CEACAM7 are similarly upregulated in both LSCC and RSCC, CEACAM18- is predominantly upregulated in proximal tumors and may serve as a biomarker for RSCC.From a clinical standpoint, several tissue (invasive), and fecal and blood (non-invasive) markers are available for diagnosis and prognosis today , 67. TheNOVA1, CELF4, and RBM20. Interestingly, NOVA1 knockdown was recently shown to significantly alter TERT transcripts in CRC [SULT2B1 (AP exon 1) observed in tumors, correlated negatively with the reduced expression of three splicing factors NOVA1, RBM20 and CELF4. Increased splice-in of CALD1 events within tumors  correlated significantly with the reduced expression of CELF4, NOVA1, and RBM20 in both left and right, suggesting differing impacts of differential regulation by these splicing factors.PTR is a key regulatory step between transcription and translation . RBPs ans in CRC . Adding FOSL1 and SP1/2/3 binding motifs as highly ranked for hypomethylated and hypermethylated genes respectively, in a side-independent manner within colon cancers. We postulate that ISX might serve as a major TF affecting hypermethylation dynamics within LSCC and RSCC. Additionally, our results indicate a positive correlation exists between epigenetic changes and gene expression of several genes, more prominently within LSCC. For instance, PHACTR3, a hypermethylated stool biomarker [Alterations in DNA methylation is an early event in cancer and has been suggested for use in non-invasive diagnosis within CRC , 59. Oveiomarker  in both In this study, we analyze multi-omics data from colon cancer patients to decipher mechanisms of tumor etiology and progression, which contribute to the side-specificity of colon cancers. We capitalize on publicly available data to identify distinctions in molecular signatures at an epigenetic (methylation), transcriptional (stage specific and stage-agnostic) and post-transcriptional levels  between right and left colon cancers. We show signatures associated with tumor aggressiveness arise from the genotoxic environment in RSCC and those with suppressed chemokine/immune response are more prevalent in LSCC. Pronounced dysregulation of miRNAs and RBPs- two well-known post-transcriptional regulators, on the right further strengthen the argument of a transcriptionally hyperactive and diverse RSCC. In addition to the differential methylation patterns, a surprising number of positive correlations  in both RSCC and LSCC underscore alternate mechanisms for expression control within colon tumors. Differences in functional mechanisms described here emphasize the molecular heterogeneity of colon cancers. Future experimental validation of the molecular players identified here, specifically within non-synchronous tumors, will influence the efficacies of existing and future diagnostic (e.g. biomarker identification), prognostic (patient stratification and recurrence) and therapeutic (e.g. molecular) interventions.Additional file 1. An excel file containing all supplementary tables highlighted with the manuscript as individual sheets.Additional file 2: Figure 1. RSCC specific miR-mRNA interaction network clustered via MCL clustering. Validated interactions between differentially expressed genes and miRs were extracted from the miR-TAR database. The network was clustered on mRNA-miRNA co-expression strength using MCL clustering. Node color: Blue- downregulated, red- upregulated, greencommonly regulated miR and orange- uniquely regulated miR. miR label color: redupregulated, blue- downregulated. Figure 2 LSCC specific miR-mRNA interaction network clustered via MCL clustering. Validated interactions between differentially expressed genes and miRs were extracted from the miR-TAR database. The network was clustered on mRNA-miRNA co-expression strength using MCL clustering. Node color: Blue- downregulated, red- upregulated, green- commonly regulated miR and orange- uniquely regulated miR. miR label color: red- upregulated, bluedownregulated. Figure 3 - Correlation network of splicing factors and AS events- the correlation network for left and right tumors captures the correlation between splicing factors  and sigAS events occurring in each side. Blue edges indicated anti-correaltion, red- correlation. Pink nodes are the splicing factors, grey nodes indicate sigAS events. The shapes correspond to each of the six events identified and presented in legend within figure.


HORVU6Hr1G087920, HORVU5Hr1G011230, HORVU5Hr1G011270 and HORVU5Hr1G011280, showed the high expression in the developing barley grains when starch accumulates rapidly. The examined 100 barley accessions could be divided into two groups based on the polymorphism of the marker S5H_29297679, with 93 accessions having allele GG and seven accessions having AA. Moreover, significantly positive correlation was found between the number of favorable alleles of the identified QTLs and TS, AC, AP content. In conclusion, the identified loci or genes in this study could be useful for genetic improvement of grains starch in barley.The content and composition of starch in cereal grains are closely related to yield. Few studies have been done on the identification of the genes or loci associated with these traits in barley. This study was conducted to identify the genes or loci controlling starch traits in barley grains, including total starch (TS), amylose (AC) and amylopectin (AP) contents. A large genotypic variation was found in all examined starch traits. GWAS analysis detected 13, 2, 10 QTLs for TS, AC and AP, respectively, and 5 of them were commonly shared by AP and TS content. qTS-3.1, qAC-6.2 and qAP-5.1 may explain the largest variation of TS, AC and AP, respectively. Four putative candidate genes, i.e.,  Hordeum vulgare L.) ranks the fourth largest cereal crop in term of planting area worldwide, only after maize, rice and wheat (FAOSTAT 2018). Like other cereals, the main composition in grains of barley is carbohydrate, mainly starch, which occupies approximately 62–77% of total dry weight , pr, prp-val10RPKM) of candidate genes. The genetic map was operated by using MapChart 2.2.0.0. .The distribution frequency, correlation and haplotype analysis, and variance analysis were conducted using IBM SPSS Statistics 25 software . The expression data were obtained from the BARLEX publicly available database and heatmap of candidate genes was drawn by TBtools  with the