Rosa hybrida) petals to better understand their involvement in petal senescence. Of 3859 proteins quantified in senescing petals, 1198 were upregulated, and 726 were downregulated during senescence. We identified 2208 ubiquitinated sites, including 384 with increased ubiquitination in 298 proteins and 1035 with decreased ubiquitination in 674 proteins. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses revealed that proteins related to peptidases in proteolysis and autophagy pathways were enriched in the proteome, suggesting that protein degradation and autophagy play important roles in petal senescence. In addition, many transporter proteins accumulated in senescing petals, and several transport processes were enriched in the ubiquitome, indicating that transport of substances is associated with petal senescence and regulated by ubiquitination. Moreover, several components of the brassinosteroid (BR) biosynthesis and signaling pathways were significantly altered at the protein and ubiquitination levels, implying that BR plays an important role in petal senescence. Our data provide a comprehensive view of rose petal senescence at the posttranslational level.Petal senescence involves numerous programmed changes in biological and biochemical processes. Ubiquitination plays a critical role in protein degradation, a hallmark of organ senescence. Therefore, we investigated changes in the proteome and ubiquitome of senescing rose ( Petunia hybrida) petals decrease dramatically during petal senescence [Hemerocallis hybrid), and iris (Iris×hollandica) [Rosa hybrida), carnation (Dianthus caryophyllus), and Tradescantia petals during senescence, causing decreased membrane integrity [Petal senescence is the terminal stage of petal development and is generally characterized by wilting, color fading, or petal abscission . Numerounescence , and a mlandica) ,6,7. Phontegrity ,8,9,10. ntegrity . Moreoventegrity ,3. HowevPetal senescence is regulated at the transcriptional, translational, and posttranslational levels ,12,13. TRose is an important ornamental plant worldwide, and flower longevity is a key indicator of commodity value. In this study, we used a label-free quantitative strategy involving antibody-based affinity enrichment and high-resolution liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis to investigate the rose proteome and ubiquitome during petal senescence. We quantified 1924 proteins and 1419 ubiquitination sites that underwent changes during rose petal senescence, providing insight into several pathways related to rose petal senescence, including proteasome and nonproteasome degradation, autophagy, hormone biosynthesis, and signaling.RhSAG12 at stages 0 to 6. The expression of RhSAG12 was barely detected at stages 0 and 1  as described previously  Figure . We then 0 and 1 . During We investigated changes in the whole proteome at stage 5 compared with stage 3. We identified 5158 proteins, of which 3859 proteins were quantified. A total of 1924 proteins showed dramatic changes in abundance with a threshold of 1.5-fold in stage 5 compared with stage 3. Among them, the expression of 1198 proteins was upregulated, while that of 726 proteins was downregulated in stage 5 compared with stage 3, respectively .To evaluate the potential functions of these differentially abundant proteins (DAPs), we performed Gene Ontology (GO) enrichment assays for DAPs a. In theIn the biological process category, DAPs were enriched in the small molecule metabolic process, organonitrogen compound biosynthetic process, organic acid metabolic process, oxoacid metabolic process, cellular amide metabolic process, carboxylic acid metabolic process, alpha-amino acid metabolic process, and carbohydrate metabolic process a. Upregup < 0.05) in stage 5 compared to stage 3, including messenger RNA biogenesis, mRNA surveillance pathway, valine, leucine and isoleucine degradation, autophagy, and metabolism of amino acids, such as histidine, tryptophan, arginine, proline, and beta-alanine. Among them, upregulated proteins were significantly enriched in glycosyltransferases, N-glycan biosynthesis, messenger RNA biogenesis, and GTP-binding proteins; downregulated proteins were significantly enriched in messenger RNA biogenesis, glyoxylate and dicarboxylate metabolism, mRNA surveillance pathway, and RNA degradation  database b. Eighteradation . These dub) sites in 1395 proteins, of which 2208 Kub sites in 1142 proteins were quantified. Among these quantified Kub sites and proteins, 384 sites in 298 proteins were identified as upregulated ubiquitination sites, and 1035 sites in 674 proteins were identified as downregulated ubiquitination sites  and wheat (Triticum aestivum) [ub. Previous studies in rice and wheat also demonstrated that most conserved ubiquitinated motifs possess alanine (A) [We used the Motif-X program to determine position-specific frequencies of amino acid residues surrounding all Kentified : 251 Kubestivum) , while tnine (A) ,30.ub sites showed increased levels of ubiquitination in stage 5 petals compared with stage 3 petals as upregulated ubiquitinated proteins, and proteins in which all Kub sites showed decreased levels of ubiquitination in stage 5 petals compared with stage 3 petals as downregulated ubiquitinated proteins. The abundance of 23 upregulated ubiquitinated proteins was decreased, including putative vesicle transport v-SNARE 12 (XP_024194691) and cytokinin signaling component AHP1 (XP_024191812)  . The abu4191812) , includi4191812) . Many ubArabidopsis , a master bZIP transcription factor in ABA signaling, ABI5, activates the expression of the major senescence-promoting NAC transcription factor ORE1 during dark-induced leaf senescence [Arabidopsis AtWRKY53 positively regulates leaf senescence by activating the expression of SAG12 and ORE9 [SAG genes [Ipomoea nil), the silencing of EPHEMERAL1, a NAC transcription factor, suppresses petal senescence [Plants have evolved complex transcriptional networks that regulate senescence; transcription factors act as master switches of transcriptional reprogramming in this process . We idennescence . Arabidoand ORE9 , while aAG genes . Membersnescence . In rosenescence ,40. In pnescence .ub sites in eight transcription factors in senescing rose petals , a leucine-rich repeat receptor-like protein kinase promotes leaf senescence by repressing cytokinin signaling and inducing auxin and ethylene signaling [Arabidopsis, mutation of AtRPK1, encoding a membrane-bound receptor protein kinase, delays age- and ABA-induced senescence [OsSIK2, encoding a receptor-like kinase, results in delayed dark-induced leaf senescence, while the sik2 mutant shows the opposite phenotype [Arabidopsis, the MKK9-MPK6 cascade plays an important role in regulating leaf senescence, with mkk9 and mpk6 mutants showing delayed leaf senescence [Zea mays), functional analysis of a member of the MPK kinase (MAPKK) family, ZmMEK1, indicated that ZmMEK1 promotes leaf senescence [Protein kinases (PKs) are essential signaling regulators of senescence . We idenignaling . In Arabnescence . In ricehenotype . In addinescence . In maiznescence .ub sites in 31 PKs were significantly changed , decreasing endoprotease activity via the cysteine protease inhibitor 2,2′-dipyridyl delays petal wilting [DCCP1 increases with the onset of ethylene production and senescence in petals, indicating that DCCP1 might be involved in ethylene-induced senescence [Alstroemeria pelegrina), daylily, iris, and petunia [We also observed that 15 putative proteases were upregulated in stage 5 compared to stage 3, including four serine proteases, six aspartyl proteases, one caspase, and four metalloproteases . In dayl wilting . In carnnescence . Moreove petunia ,15,25,56Intriguingly, the abundance of three chloroplast-localized serine proteases  and three chloroplast-localized metalloproteases  was upregulated in stage 5 compared to stage 3. In alstroemeria and iris, such chloroplast-localized proteases were also found to be associated with petal senescence ,56. It iAutophagy is one of the main mechanisms for removing and recycling useless or damaged cell components via vacuoles . The relATG genes increases during petal senescence in morning glory and petunia [An autophagy-like phenomenon has been observed in senescing leaves, with the transcription of many autophagy-related genes induced ,60. Mole petunia ,62,63. I petunia .ub sites in two enzymes participating in BR biosynthesis, DWF1  and CYP716A1 (Lys-136). Among proteins related to the BR signaling pathway, the putative receptor BRI1 (XP_024170330) was more abundant in stage 5 than in stage 3 petals  was increased at stage 5 compared to stage 3, and ubiquitination levels of Kub sites in putative BKI1  and papaya (Carica papaya), exogenous BR accelerates leaf senescence [Ubiquitination at a Kith BRI1 ; 14-3-3 ith BRI1 . These rhenotype . In mungnescence ,68. HoweS-adenosyl-met (SAM) is a precursor in the ethylene biosynthesis pathway whose formation is catalyzed by S-adenosyl-Met synthetase (SAMS). We identified five Kub sites in two putative SAMSs . In addition, SAMS (XP_024188677) was upregulated at the protein level. Petunia SAMSs show increased ubiquitination and decreased protein levels during petal senescence, suggesting possible negative feedback regulation of SAMSs by ethylene [ub sites in EIN3   . In Araband EBF2 . Taken tub sites  in JAR1 were reduced at this stage, suggesting that JA-Ile abundance is positively regulated during petal senescence via the ubiquitination pathway.In the jasmonate (JA) biosynthesis pathway, JAR1 catalyzes the conversion of JA to its active form JA-Ile . In thisArabidopsis, the application of methyl jasmonate (MeJA) accelerates leaf senescence, and genes involved in the JA biosynthesis pathway are upregulated during leaf senescence [Phalaenopsis hybrid) and Lily (Lilium L.), endogenous JA content does not change during flower senescence [In the JA signaling pathway, MYC2 activates the JA response by promoting the expression of JA-responsive genes . The effnescence . In addinescence ,74. In inescence , but in nescence .ub sites in 31 transporter proteins were significantly changed at stage 5 compared to stage 3 (ub sites in 22 proteins) were downregulated, and four of these showed greater accumulation at stage 5, including putative transporters of potassium, sucrose, lysine histidine, and molybdate-anion. Therefore, ubiquitination and de novo synthesis might jointly regulate the increase in transporter proteins during petal senescence. In morning glory, 60% of the K+ content, about 50% of the Mg2+ content, and about 25% of the Ca2+ content is transferred to other parts of the plant during petal senescence [PhPT, accumulate in senescing corollas [During petal senescence, macromolecules are degraded and remobilized in a process accompanied by enhanced transporter activities . The abu stage 3 . Intrigunescence . In petunescence ,78, and corollas . Moreovecorollas ,16,56. TArabidopsis, PTMs, including phosphorylation and ubiquitination, are involved in regulating apical-basal polarity and turnover of PINs [A putative auxin influx protein, LAX3 (XP_024166942), accumulated in stage 5 compared to stage 3. In addition, ubiquitination levels of three putative auxin efflux proteins, PIN3 , ABCB1 , and ABCB19 , were reduced during senescence . In Arab of PINs . In petu of PINs . Taken tWe performed a global investigation of the proteome and ubiquitome of senescing rose petals. Proteins related to peptidase in proteolysis and autophagy pathways were enriched in the proteome during petal senescence, suggesting that protein degradation and autophagy might play important roles in petal senescence. Many transporter proteins accumulated in senescing petals and several transport processes were enriched in the ubiquitome of senescing petals, indicating that the transport of substances is associated with petal senescence and is regulated by ubiquitination. In addition, several components of the BR biosynthesis and signaling pathways were significantly altered at the protein and ubiquitination levels, implying that BR plays an important role in rose petal senescence.Rosa hybrida ‘Samantha’) flowers were harvested at flower opening stages 0–6 [Rose  with KAPATM SYBR® FAST quantitative PCR kits (Kapa Biosystems). The expression of RhSAG12 (GenBank accession number XM_024301607) was analyzed. The rose Ubiquitin2 gene RhUBI2 was used as an internal control [Total RNA was extracted using the hot borate method as previously described , followe control . Three bg at 4°C for 10 min to remove remaining debris. The supernatant was collected, and a final concentration of 20% (v/v) trichloroacetic acid (TCA) was added. The mixtures were kept at 4 °C for 2 h and then centrifuged at 12,000× g at 4 °C for 3 min; the supernatant was discarded, and the precipitate was washed three times with precooled acetone. Finally, the precipitate was dissolved in urea (8 M), and the protein concentration was determined with a BCA kit  according to the manufacturer’s instructions.Petal samples were ground in liquid nitrogen and transferred to 5-mL centrifuge tubes. Fourfold volume of lysis buffer , deubiquitinase inhibitors PR-619 , and 2 mM EDTA was added to the cell powder, and mixtures were sonicated three times on ice using a high-intensity ultrasonic processor (Scientz). Sonicated products were centrifuged at 20,000× 4HCO3 (100 mM) to a urea concentration of <2 M. Protein samples were first digested using a 1:50 trypsin/protein ratio at 37 °C overnight and then digested using a 1:100 trypsin/protein ratio for 4 h.Dithiothreitol  was added to the protein solution and reduced for 30 min at 56 °C. Iodoacetamide  was then added and kept in darkness for 15 min at 23 °C. Protein samples were diluted with NHTryptic peptides were fractionated by high pH reverse-phase HPLC using an Agilent 300 extend C18 column  for proteome analysis and a Thermo betasil C18 column  for ubiquitome analysis. Briefly, peptides were first separated with a gradient of 8% to 32% acetonitrile (pH 9.0) over 60 min into 60 fractions. The peptides were then combined into 10 fractions (proteome assay) or 4 fractions (ubiquitome assay) and dried by vacuum freezing.Since ubiquitinated proteins have a distinct di-Gly remnant (K-ε-GG), a high-quality anti-K-ε-GG antibody (PTM Biolabs) was used to enrich ubiquitinated proteins. Tryptic peptides were dissolved in immunoprecipitation buffer . Dissolved peptides were incubated with prewashed anti-K-ε-GG antibody beads  and gently shaken at 4 °C overnight. The beads were then washed four times with immunoprecipitation buffer and twice with deionized water. Finally, the bound peptides were eluted three times, with 0.1% trifluoroacetic acid, and the eluent was dried by vacuum freezing. For LC-MS/MS analysis, the resulting peptides were desalted with C18 ZipTips (Millipore) according to the manufacturer’s instructions.Tryptic peptides were dissolved in 0.1% formic acid (solvent A) and loaded onto a reversed-phase analytical column (15-cm length and 75-μm i.d.). A gradient was achieved with solvent B (0.1% formic acid in 90% acetonitrile) increasing from 5% to 25% in 26 min, 25% to 40% in 8 min, and 25% to 80% in 3 min, and then held at 80% for the last 3 min, all at a constant flow rate of 350 nL/min on an EASY-nLC 1000 ultraperformance liquid chromatography (UPLC) system (ThermoFisher Scientific).TM Plus (Thermo) coupled online to the UPLC. The electrospray voltage applied was 2.0 kV. The m/z scan range was 350 to 1800 for a full scan. Intact peptides were detected in the Orbitrap at a resolution of 70,000. Peptides were selected for MS/MS using a normalized collision energy (NCE) setting of 28, and fragments were detected in the Orbitrap at a resolution of 17,500. A data-dependent procedure that alternated between one MS scan and 20 MS/MS scans was applied with 15.0-s dynamic exclusion. Automatic gain control was set at 5E4. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD016490.Peptides were subjected to a nanospray ionization source followed by tandem mass spectrometry (MS/MS) in a Q ExactiveRosa chinensis) genome database (https://lipm-browsers.toulouse.inra.fr/pub/RchiOBHm-V2/) concatenated with a reverse decoy database.MS/MS data were processed using the Maxquant search engine . Tandem mass spectra were searched against the rose  was adjusted to < 1%.For ubiquitinated peptides, trypsin/P was specified as the cleavage enzyme allowing up to four missing cleavages. The minimum length of peptides was set as 7, and the number of maximum labeled amino acids was set as 5. The mass tolerance for precursor ions was set as 20 ppm in the first search and 5 ppm in the main search, and the mass tolerance for fragment ions was set as 0.02 Da. Carbamidomethyl on Cys was specified as the fixed modification. Gly on Lys modification, oxidation on Met, and acetylation on the N terminus of proteins were specified as variable modifications. The label-free quantification method was LFQ, FDR was adjusted to < 1%, and the minimum score for modified peptides was set to > 40.www.http://www.ebi.ac.uk/GOA/). Proteins were classified by GO annotation into three categories: biological process, cellular compartment, and molecular function. The KEGG database was used to annotate protein pathways.Bioinformatic analysis was performed according to previously described methods ,28. Geneub sites in all protein sequences were chosen for analyzing sequence properties of ubiquitinated sites using Motif-X software (http://motif-x.med.harvard.edu/motif-x.html). All database protein sequences were used as a background database, and other parameters were set to default values. Setting parameters for motif searching using Motif-X were 20 occurrences and a Bonferroni-corrected p-value of 0.000001.Ten amino acids upstream and downstream of the K


Glucose utilization and production of chemical mediators including ATP, reactive oxygen species (ROS), nitric oxide (NO), and NADPH support effector activities of M1 macrophages. Dysbiosis is an imbalance of commensal and pathogenic bacteria and the production of microbial antigens and metabolites. It is now known that the gut microbiota-derived products induce low-grade inflammatory activation of tissue-resident macrophages and contribute to metabolic and degenerative diseases, including diabetes, obesity, metabolic syndrome, and cancer. Here, we update the potential interplay of host gut microbiome dysbiosis and metabolic diseases. We also summarize on advances on fecal therapy, probiotics, prebiotics, symbiotics, and nutrients and small molecule inhibitors of metabolic pathway enzymes as prophylactic and therapeutic agents for metabolic diseases.Maintenance of healthy human metabolism depends on a symbiotic consortium among bacteria, archaea, viruses, fungi, and host eukaryotic cells throughout the human gastrointestinal tract. Microbial communities provide the enzymatic machinery and the metabolic pathways that contribute to food digestion, xenobiotic metabolism, and production of a variety of bioactive molecules. These include vitamins, amino acids, short-chain fatty acids (SCFAs), and metabolites, which are essential for the interconnected pathways of glycolysis, the tricarboxylic acid/Krebs cycle, oxidative phosphorylation (OXPHOS), and amino acid and fatty acid metabolism. Recent studies have been elucidating how nutrients that fuel the metabolic processes impact on the ways immune cells, in particular, macrophages, respond to different stimuli under physiological and pathological conditions and become activated and acquire a specialized function. The two major inflammatory phenotypes of macrophages are controlled through differential consumption of glucose, glutamine, and oxygen. M1 phenotype is triggered by polarization signal from bacterial lipopolysaccharide (LPS) and Th1 proinflammatory cytokines such as interferon- Advanerdonkii , 8. The erdonkii –12. The The gastrointestinal (GI) tract possesses its own nervous system known as the enteric nervous system. This system communicates with the central nervous system through nerves, such as the vagus, neuromodulators, and neurotransmitters of sympathetic and parasympathetic branches of the autonomic nervous system . BacteriMicroorganisms in the gut perform their functions largely through enzyme pathways, in order to digest complex dietary carbohydrates and proteins , 14. GutBacteroides genus, and to the Clostridiaceae and Lactobacillaceae families, in special, Citrobacter and Serratia strains, produce short-chain fatty acids (SCFAs) which are volatile fat acids able to cross the blood-brain barrier via monocarboxylate transporters. SCFAs produced by intestinal bacteria are acetate (2 carbon atoms), propionate (3 carbon atoms), and butyrate (4 carbon atoms), and their molar ratios vary from 3 : 1 : 1 to 10 : 2 : 1, respectively. Most of SCFAs are metabolized to CO2. Butyrate acts on colonocytes, goblet cells, and Paneth cells and provides energy for cellular metabolism and regulates apoptosis, cellular differentiation, and chemical modification of nuclear proteins and nucleic acid. Acetate and propionate pass into the bloodstream and are taken up by the liver and peripheral organs, where they can act as substrates for gluconeogenesis and lipogenesis [β cells of the pancreas to produce insulin, whereas PYY inhibits nutrient absorption in the intestinal lumen as well as control the appetite. The gut microbiota contributes to fat deposition through the regulation of the nuclear farnesoid X receptor (FXR), the bile acid receptor that is responsible for the regulation of bile acid synthesis, and hepatic triglyceride accumulation [The maintenance of a stable, fermentative gut microbiota requires diets rich in whole plant foods, particularly rich in fibers , 14. Theogenesis . The G-pogenesis . Under cmulation . Bile acmulation . Moreovemulation . Some ofmulation .2), niacin (B3), pantothenic acid (B5), pyridoxine (B6), folate (B9), and cobalamin (B12)) that regulate the activity of host chromatin-modulating enzymes and genetic responses to environmental signals [Thousands of microbiota-derived metabolites with known and unknown functions have been identified as components of the human metabolome –21. GI m signals . Acetyl- signals . Finally signals , 24.12 of bacteria inhabits and lives in mutualistic relationships in the human GI tract [Bifidobacteria and Lactobacillus species [Bacteroides, Parabacteroides (Bacteroidetes), and Clostridium (Firmicutes) [5) and in the large intestine progressively increases in comparison with the stomach and duodenum, and the highest taxa and cell density are present in the colon, which contains 109-1012 colony-forming units per ml . They are anaerobes such Bacteroides, Porphyromonas, Bifidobacterium, Lactobacillus, and Clostridium. Anaerobic bacteria and oxygen-sensitive microbes are capable of producing of short-chain fatty acids than facultative aerobic bacteria such as E. coli, by a factor of 1000. Disruption of the dynamic interrelation between the host and the microbial communities causes dysbiosis, which is a bacterial imbalance between aerobic and facultative anaerobic bacteria ratios [E. coli and Salmonella. The rupture of gut barrier provoked by dysbiosis leads to local and systemic inflammation [Escherichia and Salmonella species [Faecalibacterium prausnitzii and Akkermansia muciniphila which predominate in healthy individuals, or by the proliferation of potentially proinflammatory bacteria such as Bacteroides and Ruminococcus gnavus, can promote the disease progression and chronicity [Akkermansia muciniphila is a mucin-degrading bacterium in the phylum of Verrucomicrobia that is present in great abundance in healthy humans, but is present in reduced number in patients with inflammatory and gastrointestinal diseases, obesity, and type 2 diabetes (T2D) [A collection of 100-400 × 10GI tract . The GI  species , 26. Alomicutes) , 26. Bacammation . A study species . Many di species –31. The ronicity –32. Compronicity . Firmicues (T2D) . In factes (T2D) .γ, antimicrobial peptides (AMPs), granulysin, defensins, lysozyme, and Reg IIIγ, which together play critical functions on the regulation of microbial ecology and immune surveillance [α-galactosylceramide, and tryptophan metabolites produce by microbial communities stimulate immune cells to produce interleukin-22, Reg3γ, IgA, and interleukin-17 [β and retinoic acid and control immune tolerance [The gut microbiota plays a critical role in the immune system by controlling the development and functionality of gut-associated lymphoid tissues (GALT), including Peyer's patches, isolated lymphoid follicles, and mesenteric lymph nodes , 36, 37.eillance , 39, 40.eukin-17 . T helpeolerance , 39. Thiolerance , 35, 36.β and IL-18. These cytokines increase the synthesis of other cytokines such as TNF-α, IL-6, IL-17, IL-22, and IL-23 and several active chemical inflammatory mediators [A variety of membrane and intracellular receptors named “pattern recognition receptors” or PRRs expressed on the epithelial and immune cells act as sensors of bacterial and cellular products, which are named the pathogen-associated molecular patterns (PAMPs) and damaged-associated molecular patterns (DAMPs) , 42. PAMediators .−/− and NOD2−/− transgenic mice have increased susceptibility not only to inflammatory bowel disease but also to type 1 diabetes and cancer [+ T cells and antibody production. Their Th17 and Treg cells are less efficient in the control of infection. Colonization of GF mice with limited number of bacterial species (gnotobiotic mouse models) can restore immunological functions [Various studies using gene-deficient mice models have defined the direct and complex interplay of bacterial dysbiosis and genetic and environmental factors , 36, 44.d cancer , 45, 46.d cancer , 48. Gerunctions , 48. Theunctions , 50. Furunctions , 48.2, NADH, and FADH2 during oxidative phosphorylation (OXPHOS) OXPHOS occurs through passing electrons along a series of carrier molecules, called the electron transport chain, with the help of electron carriers, such as NAD(P)H and FADH2, that serve as substrate to generate adenosine triphosphate (ATP) in the mitochondrial matrix [2 through three protein complexes: NADH dehydrogenase, cytochrome reductase, and cytochrome oxidase. Electron transport between the complexes occurs through other mobile electron carriers, ubiquinone and cytochrome c. The newly synthesized ATP is transported to the cytosol by adenine nucleotide translocase in exchange for ADP. Acetyl-CoA is the precursor for the synthesis of cholesterol and fatty acids, which are incorporated in the cellular plasma membranes.Mitochondria serve as the powerhouse of the cell by producing and releasing critical signals to the environment and synthesizing ATP, the body's energy required for metabolic processes . The biol matrix , 52. The+ T helper 1 (Th1), Th2, and Th17 cells, NK cells, and cytotoxic CD8+ cells [Immunometabolism comprehends the hub of biochemical activities carry out by immune cells to modulate gene expression profile and switching metabolic pathways and their key enzymes . After a8+ cells , 52. Int8+ cells .+/CD80/low/CD206high acquire and display the M1 phenotype (CD68+/CD80/low/CD206low) or M2 phenotype (CD68+/CD80high/CD206/low) after switching their metabolism and function in response to stimuli or polarization signals that initiate a pro- or anti-inflammatory response [β-oxidation of fatty acids, and mitochondrial biogenesis. In addition, M2 macrophages do not produce NO. It is important to consider that M2 macrophages display various distinct forms  depending on the local tissue environment and exposure to stimuli [Unstimulated macrophages displaying M0 phenotype and exhibiting the differentiation surface markers CD68response . M1 cellresponse –60. The response . M1 macr stimuli .γ and PGC-1β , and signaling pathways driven by AMPK (5′-adenosine monophosphate-activated protein kinase), mTORC1/2 , and STAT6  [The generation of UDP-GlcNAc intermediates promotes the glycosylation of M2-associated receptors, such as the mannose receptor . Thus, bription) , 52. A dription) .β mRNA in M1 macrophages stimulated with LPS leads to activation of the transcription factor hypoxia-induced factor 1α (HIF1α), in a glycolysis-dependent manner [α interacts with pyruvate kinase isoenzyme M2 (PKM2) and promotes the expression of HIF1α-induced genes required for glycolysis [α via ROS and increasing the expression of IL-1β [β expression, while increasing the production of immunosuppressive cytokine IL-10 [In 2013, Tannahil et al. reported that expression of IL-1t manner . HIF1α iycolysis . The actycolysis , 53, 63.of IL-1β . Inhibitne IL-10 , 63.Irg1). This enzyme converts cys-aconitate (derived from citrate) to itaconic acid. One of remarkable effect observed after itaconate treatment is the reduction of the expression of cytokines IL-1β, IL-6, and iNOS [Irg gene leads to impairment in substrate-level phosphorylation (SLP) of mitochondria [Irg 1 gene demonstrated that itaconate acts as an endogenous succinate dehydrogenase inhibitor and that such inhibition causes the accumulation of succinate [Salmonella typhimurium, Legionella pneumophila, and Mycobacterium tuberculosis; thus, it protects against infection. The cytotoxic effect is associated with the inhibition of the enzyme isocitrate lyase [Itaconate is one important metabolite formed in the second flux deviation or “break point' of the Krebs cycle pathway during macrophage transition from inactive to proinflammatory state . Itaconaand iNOS . Studieschondria . SLP is chondria . Studiesuccinate . Togethete lyase . Isocitrte lyase –68.Faecalibacterium, Bifidobacterium, Lactobacillus, Coprococcus, and Methanobrevibacter have significantly less tendency to develop metabolic disturbances and inflammation and, in turn, type 2 diabetes and ischemic cardiovascular disorders [Staphylococcus aureus and E. coli [Studies on the relationship among gut microbes, obesity, insulin resistance, and metabolic syndrome have shown an intricate interplay between host diet, genetics, and microbiome compositional dynamics , 69, 70.isorders , 73. TheA. muciniphila colonization and inflammatory conditions. In fact, A. muciniphila is present in low levels in people suffering from morbid obesity, diabetes, and cardiometabolic diseases supporting their role as antiobesity strain [Obesity is associated with behavioral and environmental factors, such as excessive consumption of energy-dense foods and sedentary lifestyle , 73–75. y strain .α receptors, which are responsible for promoting satiety via vagus nerve and dopamine release in the brain [The contribution of GI hypothalamic-pituitary-adrenal axis for predisposition to obesity is still incompletely understood , 70. Reche brain . Dopaminhe brain . RYGB ishe brain . Adminishe brain . TogetheBacillus bulgaricus increases health and prolong life span. He anticipated the rational use of probiotic made of live microorganisms with a health benefit through altering the gut microbiome [Lactobacillus plantaram and Bifidobacterium are probiotic bacteria capable of modulating negative effects of high-fat diets and even managing immunological reactions mediated by inflammatory diseases [Bifidobacterium and Lactobacillus are producers of folate in the gut. Lactobacillus rhamnosus in combination with Lactobacillus gasseri and Bifidobacterium lactis may reduce weight gain, in particular, fat tissue mass adiposity, in humans [A. muciniphila alive or pasteurized, in high-fat diet-fed mice, revealed that a small 30 kDa Amuc_1100 protein activates TLR2, allowing the development of an intestinal health immune response [The immunologist Elie Metchnikoff was the first to defend that the ingestion of fermented milk  prepared with crobiome . Lactobadiseases , 38, 45.n humans . Studiesresponse .Dietary prebiotics are known to provide immune and metabolic benefits to the host , 86. TheClostridium difficile and chronic recurrent colitis [Clostridium difficile colorectal infection after antibiotic therapy [The use of antibiotics permanently modifies gut microbial community . High do colitis . Fecal b therapy , 91. The therapy . Today, Clostridium difficile infection were cured, as compared to 31% of those receiving only vancomycin . Lactobwith FMT . Thus, fBifidobaterium infantis in mice reduces the production of proinflammatory cytokines while promoting white adipose tissue gain [A series of animal model studies have shown that the gut microbiome plays a crucial role in weight gain and obesity , 50, 70.sue gain , 71. Ressue gain . Short esue gain . Furthersue gain . Such heκB translocation [β-hydroxybutyrate  supplementation can inhibit the activity of histone deacetylases (HDACs). HDACS promote specific histone modifications to regulate transcription and DNA replication and repair. It also exerts anti-inflammatory activity by suppressing NF-κB and STAT1 activation [Acetate, propionate, and butyrate are the most important SCFAs that eventually are given as oral dietary supplementation. SCFAs acting as signaling molecules produce various physiological effects in humans . They bilocation . Butyrattivation –95. Succtivation .A. muciniphila in the fecal microbiota in obese mice [A. muciniphila is a producer of acetate and propionate, through mucin degradation, and T2D patients treated with metformin increase their butyrate- and propionate-producing bacteria, which may in turn contribute to the beneficial effect of metformin [Several small molecule inhibitors of metabolic pathways such as 2-deoxy-glucose, dichroacetate, BPTES ethyl sulfide), dimethylmalonate (DMM), rotenone, and metformin have identified as promise therapeutics , 52, 60.ese mice . A. mucietformin . It is iβ, while increasing the synthesis of immunosuppressive IL-10 [α and IL-1β, thereby promoting the reprogramming of M1 macrophages to M2 [Succinate is a strong proinflammatory mediator , 63. DMMve IL-10 . TEPP-46es to M2 . Thus, fOver the last decade, the sequencing and analyses of a large number of human microbiomes and assembly of their metabolic pathways have expanded our understanding of how bacterial metabolites participate in the microbe-host interactions in health and diseases. The functional variations of gut microbiomes among individuals have indicated the anabolic and catabolic pathways of essential importance in maintaining core community structures for whole body homeostasis. Emerging new biomarkers are promising specific discrimination of phyla and species to confirm evidences for direct participation of microbes in type II diabetes, obesity, metabolic disorders, inflammatory bowel diseases, and even certain cancers.There is no doubt that dysbiosis, by altering microbiome metabolism and consequently host metabolism, not only affects inflammatory responses and adaptive immunity but also contributes to metabolic disorders. The use of innovative pharmaceutical and nutraceutical products to manage microbial colonization and development of a healthy gut microbial community at early childhood and adult life may prevent the occurrence of common inflammatory and metabolic pathologies. Finally, the discovery and development of drugs that target enzymes of metabolic pathways, and also drive pro- and anti-inflammatory responses of immune cells, will provide the next frontier medicine for metabolic therapies in near future.


Mitochondrial fatty acid β-oxidation (FAO) is the primary pathway for fatty acid metabolism in humans, performing a key role in liver, heart and skeletal muscle energy homeostasis. FAO is particularly important during times of fasting when glucose supply is limited, providing energy for many organs and tissues, including the heart, liver and brain. Deficiencies in FAO can cause life-threatening metabolic disorders in early childhood that present with liver dysfunction, hypoglycemia, dilated hypertrophic cardiomyopathy and Reye-like Syndrome. Alternatively, FAO defects can also cause ‘milder’ adult-onset disease with exercise-induced myopathy and rhabdomyolysis. Short-chain enoyl-CoA hydratase (ECHS1) is a key FAO enzyme involved in the metabolism of fatty acyl-CoA esters. ECHS1 deficiency (ECHS1D) also causes human disease; however, the clinical manifestation is unlike most other FAO disorders. ECHS1D patients commonly present with Leigh syndrome, a lethal form of subacute necrotizing encephalomyelopathy traditionally associated with defects in oxidative phosphorylation (OXPHOS). In this article, we review the clinical, biochemical and genetic features of the ESHS1D patients described to date, and discuss the significance of the secondary OXPHOS defects associated with ECHS1D and their contribution to overall disease pathogenesis. Mitochondria are the ‘powerhouses’ of the cell, producing 95% of all cellular energy in the form of adenosine triphosphate (ATP) . ATP is 2) [2 are then oxidized by complex I (NADH: ubiquinone oxidoreductase) and complex II (succinate: ubiquinone oxidoreductase) respectively of the respiratory chain (RC) to drive ATP generation by OXPHOS. The electrons liberated from NADH and FADH2 are transferred via ubiquinone to complex III (ubiquinol: ferricytochrome c oxidoreductase), then cytochrome c, and finally complex IV (cytochrome c oxidase) which reduces O2 to generate H2O. This electron transfer facilitates the pumping of protons out of the mitochondrial matrix by complexes I, III and IV, which establishes an electrochemical membrane potential (ΔΨm) across the inner mitochondrial membrane (IMM) [m drives protons back into the mitochondrial matrix through complex V (F1F0-ATP synthase), resulting in the phosphorylation of adenosine diphosphate (ADP) to generate ATP [Under aerobic conditions, mitochondria produce ATP via three key biochemical pathways: the tricarboxylic acid (TCA) cycle, oxidative phosphorylation (OXPHOS) and fatty acid β-oxidation (FAO). Acetyl-coenzyme A (acetyl-CoA) derived from sugars, fats and proteins is oxidized via the TCA cycle to generate the reducing equivalents nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FADH2) . NADH anne (IMM) . ΔΨm drirate ATP .O-palmitoyltransferase 1 (CPT1) catalyzes the addition of carnitine to fatty acyl-CoA esters to form acylcarnitines, which are transported across the IMM via carnitine acylcarnitine translocase (CACT). Once inside the mitochondrial matrix, carnitine is removed by carnitine O-palmitoyltransferase 2 (CPT2) to regenerate fatty acyl-CoA esters and free carnitine, which is recycled back across the IMM by CACT  . Both deectively .The enzymes involved in FAO exhibit chain length specificity . Very loECHS1 gene locus was subsequently mapped to chromosome 10q26.2–q26.3 by fluorescence in situ hybridization, encoding eight exons with the 5′ and 3′ untranslated regions contained within exons I and VIII respectively [Short-chain enoyl-CoA hydratase  is responsible for the second step of FAO. ECHS1 activity was first observed by Stern and Del Campillo  in ox heectively .ECHS1 is transcribed as a single 1.4 kb mRNA, with expression observed in hepatocytes, fibroblasts and myocytes [myocytes . The tramyocytes , where tmyocytes ,14  [Defects in FAO were first described in the 1970′s in patients with carnitine ciencies ,19,20, wogenase) ,22,23. Pogenase) . Cardiacogenase) ,26. Alteogenase) .Disease presentation may not be persistent, with patients showing no symptoms or biochemical deficiencies until an episode of metabolic crisis. These episodes can be triggered by various circumstances, including prolonged fasting, exercise, infection, exposure to cold, or a fat-rich diet ,28,29. TFAO deficiencies are also believed to cause about 1–3% of unexplained sudden infant deaths , with de2 bilateral hyperintensities, a hallmark of Leigh syndrome, in almost all reported cases of ECHS1D. Despite this, there are disparities between the clinical presentation in ECHS1D and the classical features of Leigh syndrome . Indeed, et al.  proposedECHS1 variants previously associated with Leigh-like syndrome also presented with cutis laxa, a connective tissue disorder characterized by loose, inelastic skin [ECHS1 mutation , suggesting an association between this variant and PED. Interestingly, T2 hyperintensities were still observed in these patients, but Leigh-like symptoms were absent. Conversely, a sibling of one of these patients did suffer from Leigh-like syndrome with severe generalized dystonia, further exemplifying the substantial clinical heterogeneity of ECHS1D [More recently, three patients were identified with clinical symptoms that expand the phenotypic spectrum of ECHS1D ,42,43. Otic skin ,42. PED tic skin . While tECHS1 were first described in two infant siblings [ECHS1 mutations identified are missense, suggesting that null mutations may be incompatible with life [Forty-two patients (from 33 families) with ECHS1D have been described to date . As withsiblings , with alith life ,46. Otheith life ,40,42. Tith life .ECHS1 mutations are novel, the c.476A > G; p.Gln159Arg variant has been identified in seven unrelated ECHS1D patients with diverse racial backgrounds [ECHS1  has also been identified in four French-Canadian patients [While most kgrounds ,43,47,48kgrounds ,48. A seECHS1 may play a redundant role in FAO, as exemplified by the unremarkable acylcarnitine profiles in most patient cells ,49,50,52Loss of ECHS1 activity for valine metabolism also appears to play a role in disease pathology. ECHS1 acts on both methacrylyl-CoA from the valine pathway and acryloyl-CoA from an alternate pathway of odd-chain FAO that feeds into the valine pathway . In ECHSThese findings have led to tentative correlations between ECHS1D severity, degree of lactic acidosis and reduced PDC activity. In cases where ECHS1D caused early death, lactate levels were high and PDC activity low . ConversN-acetyl-S-(2-carboxypropyl)cysteine (produced from methacrylyl-CoA) is the only known metabolite that is elevated in the mildest cases of ECHS1D, making it the most useful biomarker for ECHS1D diagnosis [Other metabolic markers of ECHS1D, such as increased metabolites of methacrylyl-CoA and acryloyl-CoA in patient urine, have also been identified . In partiagnosis ,42,52.2 for oxidation by OXPHOS complexes I and II. In addition, studies have also reported physical interactions between FAO and OXPHOS proteins: OXPHOS complex I can bind the FAO protein LCHAD [FAO and OXPHOS are tightly linked biochemically, with the oxidation of fatty acids generating NADH and FADHin LCHAD , while Oin LCHAD . More rein LCHAD .Interestingly, patients with primary deficiencies in LCHAD can exhibit significant OXPHOS enzyme defects ,59. ThesSecondary OXPHOS defects have also been identified in ECHS1D. These range from isolated defects in complex I, complex III or complex IV to multiple complex I/III/IV or complex I/IV/V defects ,48,49,53While it is not clear what causes these secondary OXPHOS defects in ECHS1D, disruption of complex I activity may be explained via its interaction with the pyruvate dehydrogenase complex (PDC). As discussed above, toxic metabolites of the valine pathway can accumulate in ECHS1D and inhibit PDC activity . This inOverall, it is difficult to predict the effect of ECHS1D on OXPHOS. However, it is apparent that patients with secondary OXPHOS defects generally exhibit more severe Leigh-like symptoms .ECHS1 since the identification of the first two patients with ECHS1D in 2014. Notably, most ECHS1D patients present with Leigh syndrome or Leigh-like syndrome, a severe disorder traditionally associated with defects in OXPHOS complex I activity. While loss of ECHS1 disrupts both FAO and valine metabolism, secondary OXPHOS defects have also been identified in some patients with ECHS1D. Furthermore, these secondary OXPHOS defects are associated with a more severe clinical phenotype, suggesting that they also contribute to disease pathology.Forty patients have been described with pathogenic mutations in Like other FAO deficiencies, secondary OXPHOS defects in ECHS1D may be due to the accumulation of toxic fatty acid and/or valine metabolites, which can directly inhibit OXPHOS complex activity. Alternatively, the effects of these metabolites may be indirect by disrupting the interaction between the pyruvate dehydrogenase complex (PDC) and OXPHOS complex I. Whichever mechanism is involved, further research is required to clarify the relationship between ECHS1D and OXPHOS dysfunction, and to determine if the stability and/or biogenesis of the OXPHOS complexes is also disrupted by the loss of ECHS1. This knowledge will be invaluable for our understanding of the complex interactions between the FAO and OXPHOS pathways, and will help to advance the diagnosis and treatment of mitochondrial disorders such as ECHS1D.


Adjuvants have been used in vaccines for over a century, however, the search for safe and effective vaccine adjuvants continues. In recent decades toll-like-receptor (TLR) agonists have been investigated as potential vaccine adjuvants. In this regard, the majority of the currently investigated TLR agonists are non-protein microbial components such as lipopolysaccharides, oligonucleotides, and lipopeptides. On the other hand, a growing number of studies reveal that TLR signaling and immune responses can be activated by numerous bacterial proteins. However, their potential roles as adjuvants have been somewhat overlooked. Herein, we discuss several such bacterial proteins which exhibit adjuvant properties, including the activation of TLR signaling, antigen presenting cell maturation, pro-inflammatory cytokine production and adaptive immune response. The protein nature of these TLR agonists presents several unique features not shared by non-protein TLR agonists. These properties include the amenability for modifying the structure and function as necessary for optimal immunogenicity and minimal toxicity. Protein adjuvants can be genetically fused to protein antigens which ensure the co-delivery of adjuvant-antigen not only into the same cell but also in the same endocytic cargo, leading to more effective activation of innate and adaptive immune response. Since its discovery more than a century ago vaccines continue to save millions of lives and prevent many more from the debilitating effects of numerous infectious diseases each year . Followi+ T-cell activation, while the second necessary signal is relayed by the engagement of CD28 to CD80/CD86 , specifically related to adjuvant properties induced via TLR signaling. While searching for potential protein adjuvants we focused our attention on bacterial proteins that exhibit one or more of the following properties: (1) engage and activate TLR2 or TLR4 signaling; (2) induce pro-inflammatory cytokines; (3) up-regulate the expression of co-stimulatory molecules on APCs; (4) induce antibody mediated; or (5) induce cell-mediated immunity. We have limited our search to the TLR2, TLR4, and TLR5 agonists, because their respective receptors are expressed on the surface of APCs. Due to the cell surface expression these receptors can also be utilized to target the antigens to APCs by fusing the antigens to various TLR2/4/5 agonists.Bacterial flagellins also fall in the category of BPTAs, however, they are well-recognized TLR5 dependent vaccine adjuvants, and thus we are not discussing it further in this article. Several recent articles –31 proviTLR2 forms heterodimers with TLR1 and TLR6, which recognize tri-acylated lipoproteins and di-acylated lipoproteins respectively. Apart from lipoproteins TLR2 also respond to a diverse array of microbial patterns including peptidoglycan, lipoteicoic acid, lipoarabinomannan, zymosan, and phospholipomannan , suggestShigella flexneri (Outer membrane protein A and major outer membrane protein) (Chlamydia trachomatis (major outer membrane protein) (Shigella dysenteriae (Vibrio cholerae (OmpU) (Neisseria lactamica (PorB) (Neisseria meninigitidis (Fusobacterium nucleatum (FomA)  of protein) –49 and Cprotein)  are knowprotein) , 49, 50,protein) –50, and protein) . Bacterienteriae , 45, 65,e (OmpU) , Neissera (PorB) , Neisserigitidis , 42, andm (FomA)  have beem (FomA) , 45, actm (FomA) , 45, andm (FomA)  and humom (FomA) , 65. Panytokines .M. tuberculosis induces secretion of IL-6 and TGF-β by dendritic cells in a TLR2-dependent manner  are known to activate TLR4 signaling but not TLR2 signaling. However, DnaJ-ΔA146Ply, which is a genetic fusion of DnaJ and a ply mutant (ΔA146Ply), induces protection of mice in a TLR2-dependent manner. Furthermore, TLR2 deficiency reduces the ability of DnaJ-ΔA146Ply to induce Th1 type immune response  exhibits TLR2 and TLR4 agonist properties. Specifically, the recombinant-PepO results in a significant increase of cytokines production and neutrophils infiltration in the lungs of wild type mice compared to that of TLR2 or TLR4 knockout mice. The recombinant-PepO also induces TNF-α, IL-6, CXCL-1, and CXCL-10 in peritoneal exudate macrophages (PEMs) in a TLR2 and TLR4-dependent manner  of t manner . Moreovenfection . The recnfection . The mycnfection . S. pneuresponse . Anothert manner .LPS is the best-characterized TLR4 ligand. Upon LPS engagement the TLR4 signaling pathway results in the activation of pro-inflammatory response and maturation of APCs. In addition, TLR4 signaling by LPS also activates T-cell mediated immune response. MPLA which is a less toxic version of lipid A is being utilized as adjuvant in several vaccine formulations due to its ability to effectively activate TLR4 signaling . InteresS. pneumoniae infection , outer membrane protein-16 (Omp16) and outer membrane protein-19 (Omp19). BLS is capable of forming stable oligomers and stimulating DCs to increase the expression of CD40, CD80, CD86, and MHCII in a TLR4 dependent manner. BLS also increases the expression of several cytokines and chemokines and triggers the recruitment of DCs in vivo, depending on TLR4 signaling , heparin binding hemagglutinin (HBHA), and the 50S ribosomal protein (Rv0652) exhibit TLR4 dependent BPTA activity. RpfE, HBHA, and Rv0652 induce DC maturation by increasing the surface expression of maturation markers CD40, CD80/CD86, and MHC class I/II and the production of IL-6, IL-1β, IL-23p19, IL-12p70, and TNF-α in a TLR4 dependent manner -pulsed DCs induces a potent OVA-specific CD8+ T cell response, restrict tumor growth, and promote long-term survival  family, which interact with a variety of ligands including nucleic acids, peptidoglycan, lipo-peptides and lipids. Interaction with their corresponding ligands results in TLR dimerization, which triggers recruitment of adaptor proteins to the cytosolic Toll IL-1 receptor (TIR) domain, thereby relaying downstream signaling. Models based on the crystal structures of TLR2/1 and TLR2/6 bound to their corresponding ligands suggest that the ligands participate in forming the necessary bridge for dimerization. Two of the three lipid chains of the triacyl-lipopeptides are embedded in the TLR2 while the third one is inserted into a hydrophobic pocket of TLR1, thus facilitating dimerization of TLR2/TLR1 –70. The her TLR4 , 69. Thiher TLR4 , 69.To date, there is no model or crystal structure showing the mechanism of TLR2 and TLR4 signaling by proteinaceous ligands. TLR5 does interact with bacterial flagellin proteins, however there is no data on its mechanism of interaction. MD-2 binding to TLR4 does suggest a possible mechanism of TLR4 interaction with proteinaceous ligands. Moreover, upon looking at the LRR family proteins, it is evident that the proteinaceous ligands for this family of receptors is quite common . The conThe generation of immunity against pathogens and tolerance toward self-antigens relies on the remarkable ability of the immune system to distinguish self from non-self antigens. Pathogens first encounter the components of the innate immune system, which are endowed with germline encoded pattern recognition receptors that recognize conserved molecular patterns in microbes. Moreover, APCs employ multiple mechanisms that control any aberrant immune response against self-antigens while allowing the generation of immune response to foreign antigens , and the+ T cells (+ T cell response. Importantly, due to their protein nature BPTAs can be efficiently fused to protein antigens by genetic engineering. While antigen- adjuvant linkage cannot be achieved with many non-protein adjuvants and even require cumbersome procedures for some.At the cell surface of APCs, the TLR engagement enhances endocytosis of TLR bound ligands. While, in the endosomal compartment TLR signaling affects phagosomal maturation by enhanced acidification and fusion of the MHCII compartment. The phagosomal maturation allows the controlled proteolytic activity, thereby generating peptides from antigens associated with the TLR compartment –76. Addi T cells , 80, 81.in vitro and in vivo and induce co-stimulatory markers on macrophages and DCs. In vivo BPTAs induce recruitment of macrophages, DCs and neutrophils, which is a hallmark of adjuvant function. The APCs stimulated by BPTAs activate naïve T cells and polarize toward Th1 (BPTAs can effectively solve many problems related to the current approaches of adjuvants. As discussed above, BPTAs that activate TLR2 and TLR4 exhibit several core properties of vaccine adjuvants. BPTAs induce pro-inflammatory cytokines ward Th1 , 53, 57 ward Th1 , or Th17ward Th1 , 54. BPTward Th1 , 42, 55.ward Th1 , 57. BPTward Th1 , 54. ManSK and RS collected data related to this article. SK, RS, and EG wrote the paper. SK and EG edited the paper.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.


Targeted therapy has become a cornerstone for the treatment of melanoma patients. Targeting NRAS function is particularly challenging. To date, only single MEK inhibitor treatment was able to show minimal clinical efficacy. The discovery that co-targeting of MEK and CDK4,6 has antitumor activity created excitement for patients and clinicians; however, it is largely unknown if only NRAS mutant patients might benefit from MEK/CDK4,6 blockade.in vitro and in vivo when challenged with inhibitors of MEK, CDK4,6 and the combination of both. Data revealed, that in vitro growth response patterns of cells treated with the MEK/CDK4,6 combination correspond to in vivo efficacy of MEK/CDK4,6 co-targeting in melanoma xenograft models. Strikingly, this was consistently observed in NRAS and BRAF mutant, as well as in ‘wild type’ melanoma cells. Additionally, cells displaying elevated p-Rb levels after single MEK inhibition, showed more effective growth reduction with MEK/CDK4,6 co-targeting compared to single MEK inhibitor treatment in vivo. Findings indicate that combined MEK/CDK4,6 inhibition could offer an effectively therapeutic modality in a subset of BRAF and NRAS mutant, as well as ‘wild type’ melanoma patients.In this study we investigate response patterns of NRAS, BRAF mutant and ‘wild type’ melanoma cells  Despite recent advances, treatment of metastatic melanoma remains challenging. Inhibitors specifically targeting mutant kinases such as KIT and BRAF as well as new antibody-mediated immunotherapies have improved progression free survival (PFS) and overall survival (OS). To date, treatment decisions are based on genetic testing of mutations in a limited number of oncogenes. This approach has significantly improved patient-selection for successful kinase inhibitor treatment -4. Thus,in vitro growth response patterns using a MEK/CDK4,6 combination correlate with a response to the MEK/CDK4,6 combinatorial treatment in vivo. Additionally, induction of the cell cycle regulator p-Rb after single MEK inhibitor was found in tumors with effective co-targeting of MEK/CDK4,6 in vivo. Strikingly, results of this study reveal that this finding holds true for NRAS and BRAF mutant, as well as ‘wild type’ melanoma cells.Treatment of (N)RAS mutant tumors is particularly challenging. To date, it has been impossible to directly target mutant RAS in a clinically meaningful way and therapeutics including chemotherapy, radiation therapy and single targeted therapy using MEK inhibitors only show very limited activity once a patient has failed immunotherapy. Thus, recent findings describing that a MEK/CDK4,6 inhibitor combination is mimicking NRAS extinction in melanoma cells created excitement for physicians and patients . These rin vitro. in vitro. In MM485, WM3670 and MM415 cells the addition of the CDK4,6 inhibitor had no beneficial effect in reducing cell growth or even antagonized the effects of MEK inhibition in vitro , the CDK4,6 inhibitor PD0332991  and the combination of both inhibitors o Figure .in vitro sensitivity to MEK/CDK4,6 inhibition correlates with in vivo treatment response. Thus, we established human melanoma xenograft models using WM1366, MM415, MM485, WM3629, D04 and WM3670 cells. Reflecting current clinical treatment modalities in patients treated with small molecule inhibitors, mice received either the MEK inhibitor alone or the MEK/CDK4,6 combination. Due to the lack of activity of the CDK4,6 inhibitor in vitro, single CDK4,6i treatment was not further assessed in vivo. Control cohorts received the vehicle only. Treatment was administered by oral gavage, with the MEK inhibitor at a dosage of 2mg/kg/day and the CDK4,6 inhibitor at a dosage of 150mg/kg/day. Treatment was initiated when the tumor reached a size of about 100mm3. The MEKi alone stabilized the growth of D04, MM415, WM3670 and MM485 tumors in mice, whereas WM3629 and WM1366 tumors only showed a slight reduction of tumor size compared to mice treated with vehicle control. The combination of the MEK and CDK4,6 inhibitor was effective in mice bearing WM1366, D04 and WM3629 tumors, abolishing WM1366 tumors, significantly decreasing the size of D04 tumors and stabilizing the size of WM3629 tumors. In mice bearing MM415, MM485 and WM3670 tumors, the MEK/CDK4,6 combination did not further decrease the size of the xenograft compared to MEK inhibition alone  or the vehicle control, we extracted total protein from tumor tissue of the respective treatment groups at the end of a 3-weeks treatment cycle. Immunoblot analyses revealed largely unchanged or reduced p-ERK protein levels after MEK inhibition in WM1366 and MM485 tumors, respectively. We also noticed an induction of p-AKT in WM1366 tumors treated with the MEKi only. Interestingly, the common cell cycle downstream target, pRb, was strongly induced by the MEKi in WM1366 tumors whereas a marked reduction was noticed in MM485 tumors. The reduction in pRb in MM485 tumors was in lieu to marked induction in the pro-apoptotic marker caspase 9, whereas only a slight increase in caspase 9 was noticed in WM1366 tumors after MEK inhibition. WM1366 tumors in the mice receiving the MEK/CDK4,6 combination revealed abolished pRb levels and strong induction of caspase 9 Figure . In MM48in vitro growth response and signaling patterns in human NRAS mutant lines might also predict MEK/CDK4,6 inhibitor sensitivity of cells with different driving mutations. We incubated the human BRAF mutant cell lines A2028, A375, MM466 and Sk-Mel-28, the GNAQ mutant cell lines Mel202 and Omm1.3, the KIT mutant line WM3211, as well as C918 cells that do not harbor mutations in NRAS, BRAF, GNAQ/GNA11 and KIT  with the MEKi, CDK4,6i or the combination of both  mutant disease and to a lesser extent for KIT mutant melanoma , 2, 4, 5null mice  in 50 mM Tris-HCl (pH 8.0) supplemented with protease and phosphatase inhibitors  according to the manufacturer’s protocol. Protein concentrations were determined using the BCA Protein Assay kit . Total protein in 1x Laemmli buffer with 10% 2-mercaptoethanol was separated by SDS-PAGE, transferred 1 h to a PVDF membrane  by electro-blotting with 20% (vol/vol) methanol and blocked for 1 h in 5% (wt/vol) dry milk/TBS/0.1% (vol/vol) Tween-20. Membranes were incubated overnight at 4°C with primary antiserum following incubation with horseradish peroxidase-conjugated secondary antiserum for 1 h and developed using enhanced chemiluminescence . Phospho-ERK, phospho-AKT, cleaved caspase 9, and phospho-Rb antibodies were purchased from Cell Signaling Technologies. Beta-actin antibodies were purchased from Sigma.*106 cells of each cell line was used for subcutaneous injection in CrTac:NCr-Foxn1nu mice. Tumor size was calculated using the equation for a triaxial ellipsoid. Inhibitor treatment was initiated when tumors reached a volume of 80-100mm3. Inhibitor concentrations for single and combination therapy used in vivo were 2mg/kg/day for GSK1120212 and 150mg/kg/week for PD0332991. Compounds were administered by oral gavage fife times a week over a period of three weeks. Tumor size changes, body weight and clinical evaluations were carried out three times per week. All animal studies were approved by IACUC/LARC of the University of California San Francisco (AN086990).A total of 5-8


The intestinal microbiome is essential in humans to maintain physiological balance and nutrition metabolism. Laparoscopic cholecystectomy due to gallstone disease and cholecystitis can cause intestinal microbial dysbiosis, and following bile acid metabolism dysfunction, positions the patient at high risk of colorectal cancer. However, little is known regarding intestinal microbiota characteristics in post-cholecystectomy patients. Here, we compared the microbial composition of cholecystectomy patients with that of a healthy population. We determined that cholecystectomy eliminated aging-associated fecal commensal microbiota and further identified several bile acid metabolism-related bacteria as contributors of colorectal cancer incidence via elevation of secondary bile acids.Bacteroides, were negatively related to secondary bile acids and may be a leading cause of colorectal cancer incidence in cholecystectomy patients. Our study provides novel insight into the connection between cholecystectomy-altered gut microbiota and colorectal carcinoma, which is of value for colorectal cancer diagnosis and management.We identified aging-associated fecal microbiota in a healthy population, which was lost in cholecystectomy patients. Absent intestinal bacteria, such as  Fusobacterium nucleatum, colibactin-producing Escherichia coli, and Bacteroides fragilis can induce carcinogenesis-promoting activation of WNT-β-catenin signaling  and intestinal microbiota, thereby increasing bacterial degradation of bile acids into fecal secondary bile acids  and G protein-coupled bile acid receptor 1 (TGR5) and can influence disease occurrence and therapy  β = 0.1. Correspondingly, table of All fecal samples were collected in sterilized tubes and immediately preserved at −80°C for further analysis. The 75 healthy volunteer samples  were divided into five groups . The 60 fecal samples from 60 post-cholecystectomy patients (30–59 years old) were divided into three groups .Genomic DNA extraction was carried out using the QIAamp DNA Mini Kit . The 16S rRNA gene (V3–V4 region) amplicon sequencing was carried out with the 16S Metagenomic Sequencing Library Preparation protocol developed by Illumina . Briefly, 50 ng of bacterial DNA was amplified using primers targeting the V3–V4 variable region of the 16S rRNA gene. The 16S amplicon PCR forward primer (V3 region) was: 5′ TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG; and the 16S amplicon PCR reverse primer (V4 region) was: 5′ GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC. DNA was sequenced at Sangon Biotech  on a MiSeq sequencing instrument . The read length was centralized on 440–540 bp for the paired sequencing run.Sequencing reads were quality filtered, operational taxonomic unit (OTU) clustered. The QIIME software  was used to further analyze sequencing data , a computational approach to predict the functional composition of a metagenome using marker gene data and a database of reference genomes, uses an extended ancestral-state reconstruction algorithm to predict which gene families are present and then combines gene families to estimate the composite metagenome. Using 16S information, PICRUSt recaptures key findings from the Human Microbiome Project and accurately predicts the abundance of gene families in host-associated and environmental communities, with quantifiable uncertainty. Bray-Curtis distances were calculated using as implemented in the QIIME “beta_diversity.py” script . Community hierarchical clustering was carried out to evaluate community variation at the species level. Multiple sample comparisons were performed using Tukey-Kramer one-way analysis of variance (ANOVA). Significance was assumed for adjusted https://www.ncbi.nlm.nih.gov/sra).The sequencing data for this study can be found in the Sequence Read Archive (SRA) with accession SRP149836  identified by 16S rRNA sequencing was significantly higher in the older H4 and H5 groups than in the younger H1 group (In the oldest group (over 60 years old), abundances of operational taxonomic units including OTU1, OTU9, OTU46, OTU50, OTU57, OTU63, OTU74, OTU75, OTU91, OTU95, OTU86, OTU151, OTU153, OTU6581, and OTU12031 were significantly increased compared with that in the early stage, and showed strong positive correlation with each other. In contrast, OTU7, OTU8, OTU15, OTU27, OTU36, OTU104, OTU120, OTU201, and OTU6642 were greatly enriched and positively correlated with each other in the early stage (20–29 years old) Figure .Anaerotruncus, Akkermansia, Blautia, Faecalibacterium, and Parabacteroides in the H1 group, Akkermansia, Asteroleplasma, Faecalibacterium, Gardnerella, and Lachnospiraceae incertae sedis in the H2 group, Dorea, Faecalibacterium, Parabacteroides, Paraprevotella, Turicibacter, and Rothia in the H3 group, and Anaerotruncus, Blautia, and Lachnospiraceae incertae sedis in the H4 group demonstrated significant differences in abundances compared with the older H5 group .The above results revealed that the aging-associated intestinal microbiota was lost in cholecystectomy patients; however, greater detail on intestinal microbiota construction after significant time post-cholecystectomy (decades) is required. Thus, we compared the identified OTUs number and alpha diversity of microbiota in patients 5–10 years, 10–15 years, and more than 15 years after cholecystectomy. Results showed that, regardless of time post-cholecystectomy, patients did not exhibit remarkable variation in fecal microbiota Figures . Althouge Figure , only PsWe applied non-metric multidimensional scaling (NMDS) to distinguish fecal microbiota over the three different times post-cholecystectomy; however, no evident profiles were observed Figure . SimilarE. coli and Comamonas conversely modulate a host's response to camptothecin (CPT) and 5-fluoro-20-deoxyuridine (FUDR) management (García-González et al., Bifidobacterium, Anaerostipes, and Dorea increased in the cholecystectomy patients compared with healthy population, even the patients and healthy population had same age range (Table E. coli TOP291 increased significantly in cholecystectomy patients, suggesting cholecystectomy-related colorectal cancer could be resistant to CPT and FUDR therapies. Certainly, metabolite analysis is needed to confirm what kind of toxins is produced by these microbes in cholecystectomy patients.The human intestine is inhabited by a diverse range of microbiota, including probiotic and enterotoxigenic bacteria (Jia et al., ge Table . Some ofOscillospira species are positively correlated with secondary bile acids and negatively with primary BAs, whereas the Bacteroidetes phylum shows the opposite pattern (Keren et al., Bacteroidetes, a contributor to colorectal cancer (Keren et al., Bacteroides decreased in fecal abundance in cholecystectomy patients (Figure Mice raised under germ-free conditions show increased fecal levels of conjugated bile acids and reduce secondary bile acids in the intestines of mice, with similar bile acid profiling also revealed in inflammatory bowel disease patients (Brestoff and Artis, s Figure , suggestClostridium, Fusobacterium, Peptococcus, and Pseudomonas having the capacity to desulfate sulfonated bile acids (Gérard, Eubacterium rectale and Roseburia spp. are found at lower abundances in colorectal cancer patients (Wang et al., Sulfation can detoxify bile acid, with some genera of  Gérard, . Our seq Gérard,  because  Gérard, . FurtherWW, JW, JL, QG, and JG: study design and supervision, sample handling, data analysis, and manuscript writing. PY, RZ, and WY: sample collection and carrying out of experiments. PY and YJ: collection of samples and patient information.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.


Non-coding antisense transcripts arise from the strand opposite the sense strand. Over 70% of the human genome generates non-coding antisense transcripts while less than 2% of the genome codes for proteins. Antisense transcripts and/or the act of antisense transcription regulate gene expression and genome integrity by interfering with sense transcription and modulating histone modifications or DNA methylation. Hence, they have significant pathological and physiological relevance. Indeed, antisense transcripts were found to be associated with various diseases including cancer, diabetes, cardiac and neurodegenerative disorders, and, thus, have promising potentials for prognostic and diagnostic markers and therapeutic development. However, it is not clearly understood how antisense transcription is initiated and epigenetically regulated. Such knowledge would provide new insights into the regulation of antisense transcription, and hence disease pathogenesis with therapeutic development. The recent studies on antisense transcription initiation and its epigenetic regulation, which are limited, are discussed here. Furthermore, we concisely describe how antisense transcription/transcripts regulate gene expression and genome integrity with implications in disease pathogenesis and therapeutic development. Eukaryotic transcription of the protein-coding genes is a highly coordinated and complex process initiated by an assembly of general transcription factors and RNA Polymerase II at the promoter by an activator protein (activator), followed by elongation, and, finally, termination ,4,5,6,7.Antisense transcripts were originally identified in bacteria . Later, GAL gene cluster in yeast to study the mechanisms of antisense transcription initiation in a dextrose-containing growth medium that is permissive to antisense transcription but not to sense transcription [GAL1, GAL7, and GAL10 constitute the GAL gene cluster, a galactose-inducible genetic unit. In this cluster, GAL1 and GAL10 are divergent genes with a bidirectional promoter, while GAL10 and GAL7 are tandem genes. Such organization has significant implications in gene regulation through transcriptional interference [GAL10 coding sequence in a dextrose-containing growth medium that represses GAL10 sense transcription. Such GAL10 antisense transcription attenuates GAL1-GAL10 sense transcription. Using this system, we analyzed the mechanisms of antisense transcription initiation, from the 3′-end of the GAL10 coding sequence, in a dextrose-containing growth medium [GAL10 antisense transcription was found to be dependent on a Myb-related protein Reb1 that binds to the 3′-end of the GAL10 coding sequence [GAL10 coding sequence and 380 bp downstream of the translational stop codon [GAL10 coding sequence for histone H4 acetylation targeting TBPs (TATA-box binding proteins) and TBP-associated factors (TAFs). This forms the pre-initiation complex (PIC) in recruiting RNA polymerase II , while its sense transcription does not require TFIID (GAL10 antisense transcription does not depend on the 19S proteasome complex or 19S regulatory particle (19S RP) that is required for sense transcription  complex, is dispensable for GAL10 antisense transcription [GAL10 antisense transcription. Antisense transcription from other loci is likely to be similarly epigenetically regulated, something which needs to be elucidated further.As DNA is packaged into chromatin within nucleus, chromatin structure is likely to play an important role in regulating antisense transcription. Indeed, our recent studies demonstrated that NuA4 KAT was targeted to the antisense transcription initiation site at the 3′-end of the tylation . Such tatylation , similarn [PHO84 ,102,103.sequence ) promotehylation ,106. Thuhylation  is likelAL genes ,110,111.h medium ,96 that tor Gal4 ,89,96. Tcription ,113. HowAn antisense transcript can function by itself and/or by the act of its transcription in cis  and/or in trans (which regulates genes on other DNA strands). The trans effect is usually mediated by the antisense transcript, while the cis effect is generally due to the act of antisense transcription . Three-dHBA1) in α-thalassemia patients is repressed by antisense transcription, where an aberrant LUC7L (putative RNA-binding protein Luc7-like) RNA runs antisense to the HBA1 locus and methylates the CpG island at the promoter to repress the HBA1 gene expression [IGF2R through transcriptional interference and DNA methylation in mice [FLC (flowering locus C), namely COOLAIR (cold-assisted intronic non-coding RNA), increases histone H3 K27 methylation levels through recruitment of polycomb proteins to repress FLC expression in response to cold [FLC locus [PHO84 is upregulated upon chronological ageing and represses PHO84 sense transcription via histone deacetylation [Antisense transcription regulates sense transcription by affecting DNA methylation at the CpG islands at the promoter ,115. Forpression . Antisenpression ,117. For in mice ,119. Sen in mice . In plan to cold . In addiLC locus . In buddtylation . Furthertylation ,86,87. Itylation ,125,126.tylation ,117,127,tylation ,132,133,tylation ,134,135.Besides the functions discussed above, antisense transcription also controls mRNA splicing ,137,138,In addition to controlling sense transcription, chromatin structure, mRNA splicing and stability, translation, and generation/expression of non-coding RNAs, antisense transcription/transcripts are involved in the regulation of DNA damage response and repair. Cells are continuously attacked by genotoxic factors, and DNA lesions and damage are repaired by various cellular mechanisms, including transcription-coupled DNA repair ,155,156.CCND1  gene in response to genotoxic stress [CCND1 sense expression [CCND1 is associated with cell cycle arrest and check point activation for DNA repair. Another example is the long intergenic non-coding RNA-p21 (or lincRNA-p21) that is transcribed from the opposite strand of p21 (CDKN1A), a cell cycle regulator, in response to DNA damage [CCND1 that acts locally, lincRNA-p21 functions globally to repress transcription of the genes that are associated with apoptosis and DNA repair [INK locus is ANRIL . This is also induced by the transcription factor E2F1 (E2F transcription factor 1) in an ATM (ataxia-telangiectasia mutated)-dependent fashion in response to DNA damage [INK4A , INK4B  and ARF  [INK4A, INK4B and ARF permits cells to go back to their normal state following DNA repair, via the impediment of cell cycle checkpoints and stimulation of cell cycle progression [CDKN1A (p21) transcriptional start site. It is also induced in response to p53-dependent DNA damage [A number of studies have indicated that the expression of antisense transcripts is altered in response to DNA damage in order to control downstream gene expression for DNA repair. For example, transcription of an antisense non-coding RNA is induced from the upstream region of the c stress . Such dapression . The redA damage . Unlike A repair . AnotherA damage . The tra as p14) . Such algression . In addigression . Anothergression ,164,165.A damage . PANDA iA damage . Thus, DA damage ,168,169.HOXC locus, antisense to the HOXC11 and HOXC12 genes [HOXD locus, and silences transcription of the HOXD genes by altering the chromatin structure. Overexpression of HOTAIR induces genome-wide re-targeting of PRC2 to several hundred genes, leading to altered histone H3 K27 methylation, cancer progression and malignancy [H19/IGF2 on chromosome 11. Its overexpression is linked to cellular migration and invasion in various cancers including stomach, breast, liver, lung, and pancreas cancers [Antisense transcription/transcripts represent potential prognostic and diagnostic markers for therapeutic development for cancer. A promising candidate, HOTAIR, is significantly overexpressed in multiple tumors, including breast, colorectal, hepatocellular and pancreatic cancers ,175,176.12 genes . This an12 genes . HOTAIR lignancy ,175,176.lignancy ,175,176. cancers . Yoshimu cancers  reported cancers . MAPT-AS cancers .HOXA cluster antisense RNA 2), HOXA11-AS (HOXA11 Antisense RNA), PANDA, and ANRIL. WRAP53 regulates the tumor suppressor p53 and is overexpressed in a variety of tumor cell lines [There are other antisense RNAs that could be potential targets for cancer therapy. These include WRAP53 (WD repeat containing antisense to TP53), HOXA-AS2 , is associated with Alzheimer’s disease. BACE1-AS is a 2 kb long transcript originating from BACE1 (β secretase 1) in the antisense orientation [PINK1 antisense RNA), is transcribed from the antisense direction of the PINK1 gene that encodes PTEN (phosphatase and tensin homologue deleted on chromosome 10)-induced putative kinase 1. Mutation in the PINK locus causes the early onset of Parkinson’s disease. The PINK1-AS stabilizes the expression of a PINK1 splice variant, svPINK1, via a double strand RNA-mediated mechanism [In addition to their association with cancer, antisense transcripts are also involved in neurological disorders. The characterization of these transcripts and their modes of action may allow them to be used for diagnosis, monitoring disease progression and targeted therapies in neurological disorders. One important antisense transcript, BACE1-AS 3-9 homolog 1) to the binding site of the muscle differentiation regulator MyoD, and causes trimethylation of the histone H3 K9, thus repressing the expression of the MyoD target genes involved in muscle differentiation [MALAT1 promotes myogenic differentiation in cultured cells [MALAT1 knockout mice [+-dependent histone/protein deacetylase, SIRT1, via the formation of RNA duplexes and the facilitation of SIRT1 translation, by competing with miR34a (that can bind with SIRT1) to inhibit muscle formation [Antisense transcripts are also involved in regulating muscular diseases. For example, MALAT1 is linked to muscular dystrophy ,241. In ntiation . On the ed cells . In agreout mice . Recentlout mice . Thus, Mout mice , and theout mice ,241. Anoormation ,244. Thuormation ,245,246.ormation ,248,249.ormation . HOTAIR Since gene expression is central to cellular processes, the regulation of protein-coding gene expression by antisense transcription/transcripts has a significant impact on cellular gene expression and health. The misregulation of antisense transcription or antisense transcripts would be associated with various diseases, some of which are discussed above. However, other diseases/conditions such as aging, metabolic disorders, stress, thalassemia and spinocerebellar ataxia are also associated with antisense transcription/transcripts ,211,212.Antisense transcripts are wide-spread throughout eukaryotic genomes and are generated from independent, bidirectional or cryptic promoters. Antisense transcription/transcripts regulate gene expression and genome integrity via transcriptional interference, histone modification, and/or DNA methylation. Antisense transcripts can bring different macromolecules together within the three-dimensional context of the cell to coordinately execute transcriptional, post-transcriptional, and epigenetic processes. Thus, antisense transcription/transcripts are involved in many biological processes and are misregulated in a variety of diseases including cancer, neurological diseases, diabetes and cardiovascular disorders. Therefore, an understanding of the regulatory mechanisms of antisense transcription, characterization of antisense transcripts and their modes of actions would be useful for the diagnosis, monitoring and targeted therapies of various diseases. Indeed, several antisense oligonucleotides are in clinical trials for the treatment of various diseases . Two antAlthough antisense transcription/transcripts play crucial roles in the regulation of gene expression and genomic integrity due to their involvement in various diseases, it remains unclear how antisense transcription is initiated and epigenetically regulated. Here, we began to develop an understanding of how antisense transcription is initiated and regulated by histone covalent modifications. Further studies are needed for a thorough understanding of antisense transcription initiation and its epigenetic regulation. Such knowledge will provide new insights into the regulation of antisense transcription/transcripts and will aid in understanding the etiologies of various diseases, therefore promoting the discovery of diagnostic markers and therapeutic interventions.


A family history of pancreatic cancer (PC) is a risk factor of PC, and risk levels increase as affected families grow in number and/or develop PC at younger ages. Familial pancreatic cancer (FPC) is defined as a client having at least two PC cases in a first degree relatives. In the narrow sense, FPC does not include some inherited cancer syndromes that are known to increase the risks of PC, such as Peutz–Jeghers syndrome (PJS), hereditary pancreatitis (HP), hereditary breast ovarian cancer syndrome (HBOC), and so on. FPC accounts for 5%–10% of total PC diagnoses and is marked by several features in genetic, epidemiological, and clinicopathological findings that are similar to or distinct from conventional PC. Recent advances in genetic medicine have led to an increased ability to identify germline variants of cancer-associated genes. To date, high-risk individuals (HRIs) in many developed countries, including FPC kindreds and inherited cancer syndromes, are screened clinically to detect and treat early-stage PC. This article highlights the concept of FPC and the most recent data on its detection. Pancreatic cancer (PC) is one of the most aggressive forms of cancer in humans. The overall prognosis is quite poor, and the five-year survival rate for cases of PC is 13%. However, resection while the carcinoma is still limited to a minimal invasion (≤10 mm of tumor size) improves the five-year survival to 80% . Early PAccording to some previous case-controlled and cohort studies, it is clear that first degree relatives (FDRs) of PC patients face an increased risk of developing PC themselves  [BRCA1, BRCA2, MSH2, and MSH6 [p < 0.001). Two European FPC registries [FPC is remarkable for epidemiological characteristics relative to ordinary PC. As similar to sporadic PC (SPC) cases, smoking ,20 and d = 0.05)  characteand MSH6 ,23. Uponand MSH6 , which iand MSH6 . Anotherand MSH6 . The lifand MSH6 ,28, whilgistries ,30,31, tgistries .p < 0.05); this is a remarkable trend for incipient IPMNs (11.8-fold) [The histology of the pancreas in FPC kindreds often demonstrates multiple precancerous lesions , includi.8-fold) . The pan.8-fold) .Despite the differences in these precursor lesions ,35, a blras, TP53, and DPC4), and epigenetic  alterations frequently observed in PC [The genome-wide allelic status ,39, geneed in PC  have beeATM (variant rate: 2–4%) [BRCA1 (0–7%) [BRCA2 (4–17%) [CHEK2 (1–6%) [PALB2 (1–4%) [RAD51 (4%).Unlike other familial tumors, germline pathogenic variants have been proven in fewer than 20% of FPC cases . Recentle: 2–4%) ,43, BRCA1 (0–7%) ,45, BRCA (4–17%) ,43,46, C2 (1–6%) , PALB2 (2 (1–4%) ,49, and BRCA1/2 variants have a modest risk for PC , but other specific variants have greater increased risks. For instance, BRCA2 6174delT, a Jewish founder variant, was detected in 13% of Jewish PC cases . The BRong SPCs . A murin variant  promoted variant , which comes see  are alsoRecently, precision medicine, a new cancer treatment strategy, has been applied to the treatment of advanced cancers. One result has been that unexpected variants of cancer-associated genes are now detected as increasing rates . CommercThe notion of a FPC registry emerged with the establishment of the National Familial Pancreas Tumor Registry (NFPTR) at Johns Hopkins University , in 1994 . This waAt present, the JFPCR is planning a prospective cohort study of FPCs and their relatives; its primary goal will be to investigate the etiology of FPC and to clarify the basic and clinical issues Japanese FPCs face. Japanese experts, such as researchers, clinicians, genetic counselors, and statisticians from 21 hospitals nationwide have discussed and proposed a FPC management system. As of January 2019, 98 families and 678 HRIs had been registered with the JFPCR. JFPCR has established the expert consensus for managing the HRIs of PC (2019) and HRIs are recommended to be screened for the pancreas by the combination of image modality and blood test every six months.Worldwide experts in PC gathered at the International Symposium on Inherited Diseases of the Pancreas  and Inte2) [Several consortiums have recommended that those with a 5- ,65 to 102) .In many institutions, PC screens begin at age 40 ,74, or 1In addition to the above, many institutions recommend yearly screenings, so long as a patient’s latest pancreas image is normal . HoweverAlthough no full consensus was reached in the CAPS meeting , EUS is When investigating individuals with inherited PC, clinical images of their pancreases show a variety of characteristic findings. On the EUS, HRIs demonstrate several findings often seen in early chronic pancreatitis: cysts, high echoic foci, lobularity, strandings, and a high-echoic margin of the main pancreatic duct (MPD) . Howeverp < 0.0001) [Pancreatic investigations using computed tomography (CT), MRI, and EUS conducted in five US academic medical centers demonstrated pancreatic findings in 42% of 216 asymptomatic HRIs; these included cysts , pancreatic duct dilation (2.3%) and solid mass (1.4%), and an increasing trend by age  . A recen 0.0001) .BRCA2 and PALB2 variants) than in CDKN2A/p16 (p16-Leiden) variant carriers (42% vs. 16%). Cystic lesions were mostly stable in the FPC cohorts, while the malignant transformation of the cyst was more common (0.8% in the FPC cohort vs. 7% in the p16-Leiden cohort) [p = 0.045). However, pancreatic cysts were significantly more likely to progress in the variant carriers than in the non-carrier group  [A collaborative study by the Leiden FPC Group and FaPaCa demonstrated that pancreatic cystic lesions, or IPMNs, were frequently recognized in FPC kindreds (some of this group carried  cohort) . A simil cohort) . They re = 0.05) .STK11/LKB1 variant) [APC variant) [CFTR) [SPINK1), and cationic trypsinogen (PRSS1) [PTEN variant [IPMNs were sometimes recognized in cases with PJS (variant)  and FAP variant) . Variant) [CFTR) , serine  (PRSS1)  develop  (PRSS1) . Our pre variant . FurtherEUS-guided fine needle aspiration (EUS-FNA) and endoscopic retrograde cholangiopancreatography (ERCP) are useful in obtaining pathological evidence when abnormal findings are observed in surveillance images ,74,75. HOur diagnostic strategy is summarized in BRCA mutation, risk-reducing salpingo-oophorectomy is not only affordable, it also has an acceptable level of complications [The extent of cancer-related resections is controversial, depending on the therapeutic concept. In this context, the choices are the removal of all precancerous lesions  or the rications . Howeverications ,100 and ications . A seconications , so reseications ,103. Furp = 0.0009). In addition, all ten cases with high-grade PanIN (PanIN3) or high-grade IPMN were alive after surgery (4.1–14.7 years). These data suggest that recent surveillance systems are improving and prolonging the PC-associated survival rate among HRIs [Several surveillance results have been reported by Western FPC registries see  3,31,73,109,110.ong HRIs .Blood circulating biomarkers for detecting early pancreatic cancers have been discovered including circulating tumor DNA ,112, exoBRCA variants, superior overall survival was recognized in the cases treated by platinum-based chemotherapies than those by non-platinum agents  [p = 0.049) [BRCA-variant cases with a metastatic PC, that had not progressed during first-line platinum-based chemotherapy, demonstrated a significantly longer progression-free survival in the oraparib (poly (adenosine diphosphate-ribose) polymerase inhibitor) group (7.4 months) than in the placebo group (3.8 months) , although their overall survivals by an interim analysis were not different between the two groups  [Today, FOLFIRINOX  and gemcitabine-based regimens are standard chemotherapy regimens for unresectable PCs . In case = 0.04) . A simil= 0.049) . In a mo = 0.68) .FPCs have several characteristics that are similar to SPCs, and many others that differentiate them from the group. A family history of PC and certain genetic syndromes should be taken into consideration when screening candidates in hopes of detecting early PC. To date, scientific data regarding FPCs has been gathered via family registries. Genetic information about these HRIs can have an influence on their clinical management, and even on the treatment choice offered to them. The outcomes of HRIs’ screenings have improved in recent decades, but remain unsatisfactory. Further innovation and long-term studies are expected to detect early phase PC, the king of human cancers.