Fruits produce a range of volatile compounds that make up their characteristic aromas and contribute to their flavor. Fruit volatile compounds are mainly comprised of esters, alcohols, aldehydes, ketones, lactones, terpenoids and apocarotenoids. Many factors affect volatile composition, including the genetic makeup, degree of maturity, environmental conditions, postharvest handling and storage. There are several pathways involved in volatile biosynthesis starting from lipids, amino acids, terpenoids and carotenoids. Once the basic skeletons are produced via these pathways, the diversity of volatiles is achieved via additional modification reactions such as acylation, methylation, oxidation/reduction and cyclic ring closure. In this paper, we review the composition of fruit aroma, the characteristic aroma compounds of several representative fruits, the factors affecting aroma volatile, and the biosynthetic pathways of volatile aroma compounds. We anticipate that this review would provide some critical information for profound research on fruit aroma components and their manipulation during development and storage. Fruit quality includes both its preharvest development, such as changes in colour, flavor, and texture as fruits develop, grow, and ripen, as well as its maintenance following harvest as the perishable tissues senesce . Flavor Malaus domestica Borkh.) and peach (Prunus persica L.) [Aroma is a complex mixture of a large number of volatile compounds, whose composition is specific to species and often to the variety of fruit ,5. Althosica L.) ,8.As an important trait of fruit quality, aroma has gained increasing attention in recent years. With the fast development of science and technology especially the application of the GC-MS and other analytical apparatus, progress in aroma research has been made in several fields . In thisO-β-D-glucosides and O-diglycosides, but triglycosides have also been identified [Most fruits produce significant numbers of volatile compounds as indicators of fruit ripening. Many of these volatile compounds are produced in trace amounts, which are below the thresholds of most analytical instruments, but can be detected by human olfaction . Volatilentified . The proentified .S-methyl thiobutanoate, 3-(methylthio) propanal, 2-(methylthio) ethyl acetate, 3-(methylthio) ethyl propanoate, and 3-(methylthio) propyl acetate, also contribute to the flavor of fruit such as melons (Cucumis melo L.) [Actinidiadeliciosa) [10 monoterpenes and C15 sesquiterpenes compose the most abundant group of compounds present in the aroma profile. In some cases, these are also the key compounds determining the characteristic aroma. For example, the terpenoids S-linalool, limonene, valencene and β-pinene are key aroma compounds of strawberry (Fragaria x ananassa), koubo (Cereus peruvianus L.) and citrus (Citrus sp.) [β-ionone, geranylacetone , pseudoionone , β-cyclocitral, geranial, theaspirone, α-damascenone and β-damascenone. Their structures reveal an isoprenoid-based origin, and they were long assumed to be the products of the oxidative cleavage of carotenoids [Various types of fresh fruits produce distinct volatile profiles. Flavor volatiles are derived from an array of compounds including phytonutrients such as fatty acids, amino acids, carotenoids, phenols and terpenoids . Fruit vmelo L.) . Bound vliciosa) . Althougliciosa) . Many C1rus sp.) ,20,21. Votenoids .α-farnesene, have been proposed for cultivar classification [More than 300 volatile molecules have been reported in fresh apples . The totfication . Ethyl 2fication . For thefication . Among ffication . This hification .Pyrus pyrifolia Nak.) [E)-2-octenoate contribute to sweet or fruity odors in pears, while a high concentration of 2,4-decadienoates in fruit flesh is accepted by consumers [More than 300 volatile compounds have been identified in pear fruit (ia Nak.) , methyl ia Nak.) ,33. Otheia Nak.) . Ethyl oonsumers . In addionsumers .9 straight chain, are the major determinants of melon fruit quality perceived by consumers. These compounds are strongly dependent on the variety and physiological behavior of the fruit; in fact, fresh climacteric melons such as cantaloupe have greater aroma intensity and a shorter shelf life than less climacteric melons such as honeydew [Although the melon aroma has been extensively investigated and more than 240 volatile compounds have been identified in different varieties , the lithoneydew . In climhoneydew . Volatilhoneydew . In addihoneydew ,42. Non-honeydew . Volatilhoneydew ,45. BothH)-furanone (furaneol) and its methyl derivative 2,5-dimethyl-4-methoxy-3(2H)-furanone (mesifurane) are considered the dominating aroma compounds [γ-decalactone and 2,3-butanedione though some of these key compounds tend to be cultivar-specific [trans-2-hexenal and cis-3-hexen-1-ol are important for the green, unripe notes in strawberry aroma. Their concentrations are also cultivar and ripeness dependent [Strawberry has one of the most complex fruit aromas, made up of approximately 350 volatile compounds . The furompounds . They coompounds . Among tspecific ,48. Finaependent .E)-2-hexenal and acetoin, in “Plantain”: (E)-2-hexenal and hexanal, and in “Frayssinette”: 2,3-butanediol and two diastereomeric solerols. The most abundant aglycones were 3-methyl butanol, 3-methyl butanoic acid, solerol (two diastereomers) and acetovanillone. This compound, rarely identified in fruits, was detected for the first time in conjugated volatile compounds of fruits. The abundance of these two diastereomer in the extracts of “Frayssinette” seemed to be characteristic of this variety of banana [Although more than 250 volatile components have been identified in banana , the banf banana .3 to C4 organic acids, linalool is by far the most important alcohol. However, ketones, carvone, diacetyl, and acetoin are off-flavors. Volatile compounds in citrus fruits accumulate in oil glands of flavedo and in the oil bodies of the juice sacs. Over 100 volatile compounds have been identified for the first time in the juice of four varieties of citrus (Powell Navel orange (Citrus sinensis L.), Clemenules mandarine (C.reticulate Blanco.), Fortune mandarine (C.reticulate Blanco.) and Chandler pummelo (C.maxima Merr.)). The differences in the volatile profile in citrus juice are mainly quantitative, and only a few compounds are variety-specific. In Chandler the most characteristic volatiles were principally aliphatic aldehydes, sesquiterpenes such as nootkatone and monoterpenes such as 2-carene. Powell Navel orange showed the highest levels of esters such as nonyl acetate and of monoterpenes such as 3-carene. Clemenules showed the highest levels of ketones 3-pentanone and β-ionone and Fortune showed the highest levels of some acetate esters such as ethyl and propyl acetate, this latter almost Fortune-exclusive [α-terpineol were the most abundant among the terpenol compounds. In terms of aroma contribution to orange juice, 12 compounds were prominent based on the odour activity values (OAVs). The highest OAV values were recorded for ethyl butanoate, nootkatone, linalool and D,L-limonene [C. junos Sieb. ex Tanaka), Sudachi (Csudachi Hort. ex Shirai) and Kabosu (C.sphaerocarpa Hort. ex Tanaka), followed by γ-terpinene and β-phellandrene in Yuzu and Sudachi and by myrcene in Kabosu. Mintsulfide was newly identified in juice extracts from Yuzu and Kabosu. Among the oxygenated components, linalool was the most abundant in both the peel and juice of Yuzu and the peel of Sudachi, while both the peel and juice of Kabosu revealed the presence of high quantity of saturated aliphatic aldehydes. Wine lactone and rose oxide were identified in all the extracts, which have not been previously reported to occur in these citrus cultivars. With regard to linalool, the R-enantiomer was predominant in Yuzu and the S-enantiomer in Sudachi and Kabosu [C.natsudaidai other terpenoids are less abundant, but they exert a profound effect on aroma. These include γ-terpinene, β-phellandrene, mycerene, and α-pinene [Citrus volatiles have been extensively examined over the past several decades. Esters are important as they are responsible for the flavor characteristic .While thxclusive . Moreovelimonene . On the d Kabosu . In otheα-pinene ,56.Mangifera indica L.) possesses a very attractive flavor characteristic. More than 270 aroma volatile compounds in different mango varieties have been identified in free form [α-pinene, myrecene, α-phelladrene, σ-3-carene, p-cymene, limone and terpinolene, esters including ethyl-2-methyl propanaote, ethyl butanoate, as well as -2,6-nonadienal, (E)-2-nonenal, methyl benzoate, (E)-β-ionone, decanal, and 2,5-dimethyl-4-methoxy-3(2H)-furanone are the most important compounds contributing to mango flavor [β-ocimene, myrcene and α-terpinolene having some importance in some cultivars. Sesquiterpene hydrocarbons may also be present in amounts as high as 10% in some cultivars. There is a large variation in the quality and quantity of alcohols, ketones, and esters in mangoes, especially those of the Old World varieties. Those compounds, together with esters, are responsible for much of the characteristic aroma of Old World mangoes [Mango -3-hexenyl acetate are considered as key odorants influencing the flavor quality of peach fruit [Peach fruit volatiles have been extensively studied, and more than 100 compounds were identified ,63,64, ach fruit . Changesch fruit . Aldehydch fruit ,67.Prunus armeniaca L.) [E)-2-hexenal, linalool, 1-hexanol, ethyl octanoate, and hexyl acetate) in apricot fruit cv. “Modesto”, all of which were previously reported to be major contributors to apricot aroma [E)-2-hexenal, and their concentration decreased during ripening with significant differences. Terpenic compounds  and alcohols  were produced at a lower concentration than aldehydes, and decreased during ripening with a similar pattern to that observed for aldehydes [To date, more than 200 different volatile compounds have been described in apricots (iaca L.) . Six majot aroma ,70. The ldehydes .Vitis vinifera L.) volatiles include a great number of compounds, among which monoterpenes, C13 norisoprenoids, alcohols, esters and carbonyls are found [trans-furan linalool oxide , cis-furan linalool oxide , benzyl alcohol and 2-phenylethanol. Other monoterpenes potentially contributing to Muscat aroma were rose oxide, citral, geraniol, nerol and citronellol [E)-2-hexenal was the most abundant volatile compound in Riesling and Cabernet Sauvignon grapes, and it showed a significant increase in concentration after veraison. Benzene derivatives discriminated ripe Cabernet Sauvignon grapes, whereas monoterpenes and sesquiterpenes discriminated both cultivars pre-veraison with a broader range of terpenes observed in the Cabernet Sauvignon samples compared with the Riesling samples. At veraison, terpene production in both varieties was low, but Riesling grapes produced some terpenes (geraniol and α-muurolene) post-veraison. Generally, esters and aldehydes were the major class of compounds from Riesling grapes, while Cabernet Sauvignon showed a greater tendency to form alcohols [Grape (re found . Grape mre found . Octanoire found . In addire found . The mosronellol . (E)-2-halcohols .Rubus idaeus x ursinus) have been studied extensively. At least 200 volatile compounds have been identified in this fruit. Many compounds including raspberry ketone, α-ionone, β-ionone, linalool, (Z)-3-hexenol, geraniol, nerol, α-terpineol, Furaneol, hexanal, β-ocimene, 1-octanol, β-pinene, β-damascenone, ethyl 2-methylpropanoate, (E)-2-hexenal, heptanal, and benzaldehyde have been identified to contribute raspberry aroma. Among them, α-ionone, β-ionone, geraniol, nerol, linalool, and raspberry ketone could be particularly important to red raspberry aroma [α-ionone, β-ionone, geraniol, linalool, and (Z)-3-hexenol were observed in different raspberry cultivars. In addition, the volatile compositions in “Meeker” raspberry grown at different locations also varied [Rubus laciniata) has been widely planted, the study of blackberry flavor is still very limited. The early studies focused on the volatile constituents of blackberry, and very diverse compounds have been identified. 2-Heptanol, p-cymen-8-ol, 2-heptanone, 1-hexanol, α-terpineol, pulegone, 1-octanol, isoborneol, myrtenol, 4-terpineol, carvone, elemicine, and nonanal have all been identified as the major volatiles [β-ionone, and hexanal could be most important in “Marion”, while in “Black Diamond”, the most important compounds were linalool, β-ionone, furaneol, and 2-heptanol. The major difference between the cultivars for aroma compounds was that “Marion” had higher OAV of furaneol than “Black Diamond”, while “Black Diamond” had higher OAV of linalool than “Marion”. The chemical analysis results matched with the descriptive sensory evaluation that “Marion” had more berry, fruity, strawberry aroma while “Black Diamond” had more floral aroma [Aroma compounds in raspberry cultivars  , twelve of these volatiles are reported for the first time in highbush blueberries, with 10 being positively identified: (Z)-3-hexenal, -2,4-hexadienal, -2,6-nonadienal, -2,4-nonedienal, methyl 2-methylbutanoate, butyl acetate, 2-methylbutyl acetate, and geranyl acetate. “Primadonna” was characterized by a large amount of esters and C6 aldehydes. In contrast, fewer than four esters were found in “FL02-40” and “Snowchaser”, respectively, but they produced more terpenoids than “Primadonna” and “Jewel” [A total of 42 volatiles were identified in four southern highbush blueberry ( “Jewel” .Ananas comosus L. Merr.) [δ-octalactone. The OAVs of five compounds including ethyl-2-methylbutyrate, methyl-2-methylbutyrate, 3-(methylthio) propanoic acid ethyl ester, ethyl hexanoate and decanal were considered to be the characteristic aroma compounds for the Tainong No. 6 pineapple [H)-furanone (DMHF), decanal, ethyl 3-(methylthio) propionate, ethyl butanoate, and (E)-3-ethyl hexenoate; while in core the main compounds were ethyl-2-methyl butanoate, ethyl hexanoate and DMHF. The highest odor units were found to correspond to ethyl 2-methyl butanoate, followed by ethyl hexanoate and DMHF [More than 280 volatile compounds have been found in pineapple fruit (. Merr.) . Esters . Merr.) . A totalineapple . The chaand DMHF .Z)- and (E)-2-hexenal, hexanal, (Z)- and (E)-3-hexenol, and methyl benzoate [More than 80 compounds related to fruit aroma have been identified in kiwifruit, with the major components being methyl and ethyl butanoate, -2-hexenal, acetic acid, butyl ester, acetic acid, hexyl ester, etc., representing 93.81% of the total aromatic content. β-Damascenone and estragole in “Zaofengtian”, which weren’t detected in “Vista Bella” or “Liaofu”, could be unique components to “Zaofengtian” [Evaluation of volatiles at the germplasm level is useful for future breeding efforts, aimed at improvement of fruit quality, via effects on fruit aroma. The composition and concentration of grape volatiles largely varied with genetic background. In grape cultivars belonging to seven genotypic groups, Cmurensis . The maiengtian” . In straengtian” ,90.rin gene to reduce ethylene production and slow tomato fruit softening, resulted in some deterioration in flavor quality and reduction in flavor volatiles [Insertion of the olatiles . Transgeolatiles . Fruit wolatiles . The traolatiles .Many factors including cultivar, cultural practices, ripeness, harvest maturity and postharvest handling can influence the abundance of volatile compounds in fruit. Of these factors, maturity is one of the critical factors to influence the abundance of volatile compounds in fruit . Ideally6 aldehydes and alcohols products of the enzymatic breakdown of unsaturated fatty acids, are major contributors to the flavor of immature fruits in the absence of furanones and esters. During fruit ripening, levels of C6 compounds decrease drastically with increasd furanone acid lactone and ester production [6 aldehydes were identified as the major compounds in immature white fruit, while furanone and esters are present in three quarters or fully red fruit [Z)-3-hexanol and (E)-2-hexenal, the most abundant volatiles in whole green and ripe “Fuerte” avocados (Persea Americana Mill), declined with ripening, while overripe fruit had the highest ester concentration [E)-2-hexenal and 2,4-hexadienal greatly declined in amount, while acetaldehyde, methyl acetate, pentanal, and β-myrcene were at higher concentrations in mature fruit and may also have contributed to the overall flavor [et al. [6 compounds and terpenoids, were evident before veraison in three different flavour table-grapes, “Jingxiu”, “Bimeijia” and “Jingya”, while most of the esters were detected at or after veraison. C6 compounds increased in the early period of maturation, and then decreased. Most alcohols and carbonyls tended to continuously decrease during ripening. Except for geraniol, terpenoids increased until maturation, then decreased. Some esters continued to increase after maturation. Principal component analysis showed that terpenoids and esters were the characteristic volatiles of ripe “Bimeijia” and “Jingya” grapes, respectively. “Bimeijia” had the highest terpenoid content at maturity, while “Jingya” continued to accumulate some esters after maturation.In apples, immature fruits produce low quantities of volatiles at harvest, and lose the capability of volatile production during storage more readily than mature fruit . In “Cigoduction . The enhoduction . Similared fruit . Flavor ed fruit . El-Magentration . Pereirantration . During l flavor . In detal flavor . Yang et [et al.  reportedSolanum lycopersicum L.). Grape aroma potential was highest in vines under mild water deficit and moderate nitrogen supply. Severe water deficit stress seemed to limit aroma potential, as did nitrogen deficiency [Pre-harvest factors such as sunlight, water availability, fertilization, and chemical applications affect crop growth, and can affect internal quality characteristics of the harvested product, including flavor. Heavy rains prior to harvest dilute flavor compounds in tomatoes  and ADH enzyme activities in the oxylipin pathway. Heating-inhibition of C6 volatile production, however, does not appear to be due to down-regulation of gene expression, but HPL and ADH activities were briefly suppressed [Storage temperature is a fundamental factor affecting the flavor of fruits. Changes in storage temperatures had no major effects on aroma volatile contents in chilling-tolerant “rivative . Productrivative . Refrigeactivity . During activity . Both chppressed .2 and raising CO2 can maintain the quality of many fresh fruits for extended periods. However, exposure of fresh product to O2 levels below their tolerance level can increase anaerobic respiration and lead to the development of off-flavor. Storage of fruit under controlled atmosphere (CA) conditions can reduce the capacity of several fruit to produce ethylene and alter production of aroma volatiles [2 + 10% CO2 at 2 °C, improved juiciness, sweetness, perception of flavor, emission of aroma volatile compounds and sensory acceptance in comparison with fruit stored in cold air [2 levels and increasing CO2 levels of “Titania” blackcurrants retarded the capacity of 3-week stored fruit to synthesize terpenes. Differential changes among the various groups of terpenes were more important, where terpene alcohols reached a peak in 6-week air-stored fruit, and storing berries under a high CO2 level (18 kPa) and/or decreasing O2 level (2 kPa) resulted in lower biosynthesis of these alcohols compared to control fruit. CA storage conditions led to a transitory reduction in the emission of alcohols but a recovery was recorded with prolonged storage. Non-terpene esters differed greatly in storage, in particular the ester ethyl butanoate. Air-stored fruit at both sampling dates synthesized significantly higher amounts of esters than freshly harvested fruit but a significant decline was observed for branched butyl substances (2-methyl butyl butanoate) after 6 weeks storage [Lowering Oolatiles . Storagecold air . Decreas storage .2 and elevated CO2 levels, similar to that of CA. Use of edible coatings affects flavor and the level of volatile flavor compounds in citrus, apple and mango fruit [A. vera gel (1:1 or 100%) slightly delayed fruit ripening but reduced fruit aroma volatile development. A. vera gel coating did not exceed the commercial mango “carnauba” and Semperfresh in retarding fruit ripening and improving aroma volatile biosynthesis [Use of packaging and edible coatings can create a modified atmosphere (MA) with reduced Ogo fruit ,113,114.go fruit . The effIn addition to CA, other gaseous treatments of fruits and vegetable have been reported. Use of ethylene to synchronize ripening has been practiced for years on banana and tomato, and for degreening of citrus. Ethylene treatment of tomato fruit alters volatile levels . 1-methyOther chemical treatments of fresh product may also affect flavor. Calcium treatment of fruit is a widely used practice aimed mainly at avoiding the development of bitter pit. Calcium treatment of commercially mature “Golden Reinders” apples notably enhanced the production of aroma volatile compounds after mid-term storage under air and, to a lesser extent, under standard CA. Aroma volatile production was severely depleted in ultra-low oxygen atmosphere stored samples, and calcium treatment could not overcome this inhibition . Methyl As volatiles are comprised of at least five chemical classes, there are several pathways involved in volatile biosynthesis. These have not been fully described but appear to be common for different fruits. Volatiles important for aroma and flavor are biosynthesized from amino acids, membrane lipids and carbohydrates . Althoug1 to C20 are important character-impact aroma compounds that are responsible for fresh fruit flavors with high concentrations, and are basically formed by three processes: α-oxidation, β-oxidation and the lipoxygenase pathway [β-oxidation biosynthetic pathway, whereas when fruit tissue is disrupted, volatiles are formed via the lipoxygenase (LOX) pathway [β-oxidation [Fatty acids are major precursors of aroma volatiles in most fruit . Fatty a pathway . Aroma v pathway . Neverthxidation . Many ofxidation . In addixidation . Autoxidxidation . Hexanalxidation .β-oxidation is a major process for the formation of flavor molecules in all organisms, the specific pathways in plants are not well understood. Baker et al. described varied roles for this pathway in relation, not only to fatty acid catabolism but also, to amino acid metabolism and biosynthesis of hormonal compounds [β-Oxidation results in successive removal of C2 units (acetyl CoA) from the parent fatty acid. The detailed mechanisms of conventional β-oxidation are well established [et al. reported that β-oxidation of fatty acids is the primary biosynthetic process providing alcohols and acyl coenzyme A (CoAs) for ester formation [β-oxidation cycle, requiring flavinadenine dinucleotide (FAD), nicotinamide adenine dinucleotide (NAD), and free CoA. Acyl CoAs are reduced by acyl CoA reductase to aldehyde that in turn is reduced by ADH to alcohol for use by AAT to produce esters [β-oxidation pathway [Although the degradation of straight chain fatty acids by α and ompounds . β-Oxidaablished . Sanz etormation . Fatty ae esters . Pear an pathway .Cocos nucifera L.) , is also associated with the β-oxidation pathway [β-oxidation, and LOX [The biosynthesis of lactones, key aroma components in fruits such as peach and nectarine , pineapple , or coconut -enal isomerase and ADH. When fruit are homogenised, linoleic and linolenic acid are oxidised to various C6 and C9 aldehydes [The metabolism of polyunsaturated fatty acids, via the first LOX-catalyzed step and the subsequent reactions, is commonly known as the LOX pathway. Saturated and unsaturated volatile Csistance ,137. At ldehydes . In intaldehydes . During ldehydes . The LOXldehydes . If the trans-2-hexenal, cis-3-hexenol and methyl jasmonate are derived from C18 unsaturated fatty acids including linoleic acid or linolenic acid, which undergo dioxygenation in a reaction catalyzed by LOX [9 or C13 positions yielding two groups of compounds, the 9-hydroperoxy and the 13-hydroperoxy derivatives of polyenoic fatty acids. These derivatives can be further metabolized by an array of enzymes, including allene oxide synthase (AOS) and HPL, which represent two branches of the lipoxygenase pathway yielding volatile compounds. In the AOS branch of the lipoxygenase pathway, 13-hydroxyperoxy linolenic acid is converted to 12,13-epoxyoctadecatrienoic acid by AOS [6 or C9 volatile aldehydes  and the corresponding C12 or C9ω-fatty acids . HPL C6 aldehyde products can be further converted to their isomers by spontaneous rearrangement by alkenal isomerases, or reduced to alcohols by the action of ADH [As shown in d by LOX . These ed by AOS . A seried by AOS . In the n of ADH .α-keto acid , monoterpenes (C10), sesquiterpenes (C15), homoterpenes (C11 and C16), and some diterpenes (C20) have a high vapor pressure allowing their release into the atmosphere. As shown in 5 precursor isopentenyl diphosphate (IPP) and its allylic isomer dimethylallyl diphosphate (DMAPP), which in higher plants are generated from two independent pathways located in separate intracellular compartments. In cytosol, IPP is derived from the long-known mevalonic acid (MVA) pathway that starts with the condensation of acetyl-CoA [The terpenoids compose the largest class of plant secondary metabolites with many volatile representatives. Hemiterpenes , the natural precursor of sesquiterpenes [10), the universal precursor of all the monoterpenes [20 diphosphate precursor of diterpenes [In plastids, DMAPP generated from the MEP pathway is used by isoprene synthases for isoprene formation ,156. In terpenes . In plasterpenes . The conterpenes ,159. Folterpenes ,161. Oneterpenes .trans-isopiperitenol, a volatile compound found in mint (Mentha aquatic) [trans-carveol, which undergoes further oxidation by nonspecific dehydrogenases to form carvone, a major aroma volatile of caraway fruits (Carum carvi L.) [S-citronellol, the precursor of the potent odorant rose oxide, was confirmed enzymatically in grape mesocarp [16 and C11 carbon skeletons, so called homoterpenes, which are mainly emitted from injured tissues. Although the exact biosynthetic routes to homoterpenes, -TMTT (C16) and (E)-DMNT (C11) still remain unclear, it is believed that they are derived from geranyl-linalool (C20) and (3S)-(E)-nerolidol (C15), respectively, by oxidative degradation possibly catalyzed by cytochrome P450 enzymes [Many of the terpene volatiles are direct products of terpene synthases, while others are formed through alterations of the primary terpene skeletons made by TPSs by hydroxylation, dehydrogenation, acylation, and other reaction types . 3-Hydroaquatic) , while 6arvi L.) ,165. Anoarvi L.) . On the arvi L.) ,168. Recmesocarp . Modific enzymes .β-ionone in Arabidopsis  [Solanum lycopersicum) and petunia (Petunia hybridea) [Carotenoid and their apocarotenoid derivatives are isoprenoid molecules important for the primary and secondary metabolisms of plants and other living organisms . The oxihaliana) , tomato ybridea) ,176.AtCCD1 cleaves linear and cyclic carotenoids at the 9,10 and 9′,10′ positions. For example, when β-carotene serves as the substrate, AtCCD1 produces two C13 products (both β-ionone) and a central C14 dialdehyde  and a C27 aldehyde . CCD8 can then cleave the C27 aldehyde at its 13,14 double bond [BoLCD from Bixa orellana was reported to cleave at the lycopene 5,6 double bond [CsZCD from Crocus sativus was found to cleave at the 7,8 double bond of zeaxanthin [CCDs exhibit specificity for the double bond that they cleave but many are promiscuous in their substrate choice. In plants, CCD1 and CCD7 cleave the 9,10 double bonds of their respective carotenoid substrates, aldehyde . CCD7 cable bond . Subsequble bond . In addible bond , and CsZaxanthin .Fruit aroma is an important indicator to reflects the quality of fruit flavor. Fruits synthesize and emit a large variety of aroma volatile compounds with terpenoids and fatty-acid derivatives the dominant classes. Whereas some volatiles are probably common to almost all fruits, others are specific to only one or a few related fruits. Production of volatiles was markedly influenced by many factors, to date we have a limited understanding of how these factors interact to determine the actual volatile composition and resulting flavor of the fruit. In general, more than one biochemical pathway is responsible for a blend of volatile compounds released from different fruits. Within this final issue, further information about the genes that are involved in the synthesis of aroma volatile should be reviewed.


Bordetella pertussis and the edema toxin (ET) of Bacillus anthracis are both calmodulin-activated toxins with adenylyl cyclase activity that invade host cells and massively increase the cellular concentrations of a key second messenger molecule, 3’,5’-cyclic adenosine monophosphate (cAMP). However, the two toxins differ in the kinetics and mode of cell entry and generate different cAMP concentration gradients within the cell. While CyaA rapidly penetrates cells directly across their plasma membrane, the cellular entry of ET depends on receptor-mediated endocytosis and translocation of the enzymatic subunit across the endosomal membrane. We show that CyaA-generated membrane-proximal cAMP gradient strongly inhibits the activation and phosphorylation of Syk, Vav, and Pyk2, thus inhibiting opsonophagocytosis. By contrast, at similar overall cellular cAMP levels, the ET-generated perinuclear cAMP gradient poorly inhibits the activation and phosphorylation of these signaling proteins. Hence, differences in spatiotemporal distribution of cAMP produced by the two adenylyl cyclase toxins differentially affect the opsonophagocytic signaling in myeloid phagocytes.Myeloid phagocytes have evolved to rapidly recognize invading pathogens and clear them through opsonophagocytic killing. The adenylate cyclase toxin (CyaA) of  Phagocytosis is a central event in the innate immune response and is defined as the mechanism for recognition and internalization of particles larger than 0.5 µm in diameter ,2. It isMβ2, Mac-1), which recognizes particles coated with the opsonizing proteins of the complement system and triggers signal transduction through RhoA, leading to the sinking of the opsonized particle into the cell [Monocytes, macrophages, neutrophils, and dendritic cells express a variety of receptors that mediate (opsono)phagocytosis. Type I phagocytosis occurs through the Fcγ receptor (FcγR) that binds particles opsonized by antibodies. This triggers signal transduction through small guanosine triphosphatases (GTPases) of the Rho family, Cdc42, and Rac, leading to engulfment of the opsonized particle through protruding lamellopodia ,4,5. Typthe cell ,4,5. CR3the cell ,7. Phagothe cell ,9, the pthe cell ,11, and the cell ,13,14, wMany bacterial pathogens secrete exotoxins that inhibit the bactericidal activity of phagocytes by specifically targeting the signaling pathways involved in the uptake of invading pathogens. A prominent class of such virulence factors are the toxins with adenylyl cyclase (AC) enzyme activity ,16,17,18Bordetella pertussis secretes an adenylate cyclase toxin (CyaA) [+ efflux from cells [The gram-negative respiratory pathogen n (CyaA) , which in (CyaA) . CyaA bin (CyaA) . The haln (CyaA)  and insin (CyaA) ,29. In pom cells .Bacillus anthracis that secretes an A-B type edema toxin (ET) consisting of protective antigen (PA) and edema factor (EF) [A structurally homologous AC enzyme is produced by the Gram-positive zoonotic pathogen tor (EF) . PA usestor (EF) . Upon cetor (EF) . Within tor (EF) ,31.The kinetics and mode of entry of CyaA and ET into target cells differ, thus yielding cAMP production in different zones of the cell cytosol . CyaA deIt was previously shown that CyaA-mediated cAMP signaling swiftly inhibits the bactericidal activity of phagocytes by modulating the activity of key components of the signaling cascades involved, such as Syk, RhoA, and SHP-1 ,34,35,36We first compared the time course of cAMP production in THP-1 cells exposed to CyaA or ET, in order to define a time point at which comparable overall levels of cellular cAMP were reached. Towards this aim, the toxins were incubated with human THP-1 monocytes that express the cellular receptors for both toxins ,37,38. AIn contrast, even at a higher molar concentration, ET  triggered a delayed and relatively slow rise of cAMP levels over the initial four hours, followed by a rapid increase of cAMP level B. This cCyaA- and ET-provoked cAMP elevation hijacks signal transduction pathways and interferes with the opsonophagocytosis and bactericidal activities of phagocytes ,35,36,40To trigger the opsonin receptor-activated signaling pathways involved in opsonophagocytosis ,36, THP-Intracellular signaling mediators Syk, Pyk2, and Vav are crucial for signal transduction during phagocytosis of opsonized targets ,2. GivenThe non-receptor tyrosine kinase Syk is recruited by the immunoreceptor tyrosine-based activation motifs (ITAMs) and is phosphorylated (activated) in the course of opsonophagocytosis, forming a multi-molecular signaling complex in which Syk-mediated phosphorylation of several adaptor proteins promotes activation of key signal transduction pathways ,9,11. AsTo corroborate this observation, we next examined the activation of the cytoplasmic non-receptor tyrosine kinase Pyk2, which is activated by signaling through phagocytic receptors or upon increase of cytosolic calcium concentration. Pyk2 activation is downstream to activation of the Syk and Src kinases and regulates the Rho/WASP pathway of actin-polymerization ,11,42,43Finally, we corroborated the above observations by examining activation of the Rho family guanine nucleotide exchange factor Vav, which plays a crucial role in the transmission of signals from the phagocytic receptors to Rho GTPases during phagocytosis. Syk physically interacts with Vav downstream of ITAMs and phosphorylates Vav on tyrosine-174 ,12,14,44Hence, CyaA-produced membrane-proximal cAMP intoxication  in monocAdefovir diphosphate, a cellular metabolite of the clinically approved pro-drug adefovir dipivoxil, was shown to competitively and selectively inhibit the AC enzyme activities of EF (Ki = 27 nM) and of CyaA (Ki = 25 nM) . Therefo2+ influx, K+ efflux, and pore formation in the cellular membrane, do not affect the SOZ-induced tyrosine phosphorylation of Syk, Pyk2, and Vav.At the same time, adefovir dipivoxil itself did not trigger any changes in tyrosine phosphorylation of Syk, Pyk2, and Vav C–E or inEngagement of phagocytic receptors triggers downstream signaling and tyrosine phosphorylation of crucial signaling proteins, provoking actin cytoskeleton rearrangement and uptake of opsonized targets ,10,12,13Phagocytosis is a multistep process that involves receptor-mediated interaction and recognition of phagocytic targets, actin cytoskeleton remodeling facilitating uptake, phagosomal membrane remodeling, phagosome maturation, and finally, the clearance of the ingested particles. A variety of signal transduction pathways contribute to each step of the carefully orchestrated and tightly controlled cellular phagocytic process ,2. Parti2+ channel Cav1.2 is influenced by intrinsic AC activity through cAMP-mediated activation of PKA, which is counterbalanced by the activity of the phosphatase PP2A, and all of these enzymes assemble into a membrane-proximal multi-molecular signaling complex [B. anthracis to the lymph nodes, from where the replicating bacteria can disseminate systemically [In this study, we demonstrated that CyaA-mediated cAMP elevation, but not ET-mediated cAMP elevation to comparable overall levels, triggers signaling events that inhibit the uptake of opsonized targets . CyaA-tr complex . It was  complex . SHP-1 a complex  and enab complex ,29. Howeemically . ImportacAMP signaling can be highly compartmentalized. It is heavily influenced by the cell specific expression of PKA isozymes, the expression and specific subcellular localization of A-kinase anchoring proteins , the distribution and localization of Epac, and the localization and regulation of phosphodiesterases  ,55,56,57B. anthracis Ames spores and fluorescent E. coli particles) in differentiated human macrophages [ET-mediated cAMP accumulation, with a gradient radiating from the perinuclear region, was shown to provoke IL-6 secretion and inhibit LPS-induced TNF-α secretion in bone marrow derived-macrophages , paralyzrophages . Howeverrophages ,7,59.Taken together, this study shows that the spatial distribution of toxin-provoked cAMP intoxication in phagocytes governs the inhibition of particle uptake and phosphorylation/activation of important signaling proteins. This reveals that mere accumulation of supraphysiological amounts of cAMP in the cytoplasm is not sufficient for effective modulation of some of the signaling pathways involved in opsonophagocytic particle uptake and that appropriate spatial distribution of cAMP is key to effective inhibition of opsonophagocytic uptake by the adenylyl cyclase enzyme toxins.2, 2 mM MgCl2, 1% (w/v) glucose, and 1% (v/v) fetal calf serum (FCS)) was prepared for flow cytometry experiments. Zymosan A was purchased from Sigma-Aldrich, St. Louis, MO. For labeling, zymosan at 10 mg/ml was incubated with 50 ug/ml of fluorescein isothiocyanate  in 300 mM sodium bicarbonate buffer (pH 9.2) with overnight rotation at 4 °C. Unbound FITC was removed by extensive washing with HBSS buffer, and FITC labeled zymosan (FITC-zymosan) was stored at −20 °C.RPMI 1640 and DMEM were obtained from Sigma-Aldrich, St. Louis, MO. Antibodies against p-PYK2 (sc-293142), p-Vav (sc-16408-R), and Vav (sc-17831) were obtained from Santa Cruz Biotechnology Inc., Dallas, TX. Anti-phosphotyrosine (11-263-C100) and anti-Syk (11-376-C100) antibodies were purchased from Exbio, Vestec, Czech Republic. Anti-Pyk2 antibody (P3902) was purchased from Sigma-Aldrich, St. Louis, MO. Antibody against p-Syk (MAB6459) was purchased from R&D systems, Minneapolis, MN. Rabbit polyclonal anti-cAMP antibody for competitive ELISA was obtained from GenScript, Piscataway, NJ. Horseradish peroxidase (HRP)-conjugated anti-mouse and anti-rabbit IgG antibodies were purchased from GE Healthcare, Piscataway, NJ. HBSS buffer  complemented with 2 mM CaClE. coli strain XL1-Blue . The protein was chromatographically purified by ion exchange chromatography on DEAE-Sepharose and hydrophobic chromatography on Phenyl-Sepharose, from which endotoxin was removed by repeated washing of the toxin-bound resin with 60% isopropanol [2 buffer at ‒20 °C. Recombinant EF and PA from Bacillus anthracis were purchased from List Biological Laboratories, Inc., Campbell, CA.CyaA was produced in the presence of activating acyltransferase CyaC in propanol ,60. The THP-1 human monocytic cells (ATCC TIB-202) were obtained from the American Type Culture Collection  and tested for the absence of mycoplasma contamination. The cells were cultured in RPMI 1640 supplemented with 10% fetal calf serum and antibiotic antimycotic solution. The cells were transferred to DMEM with 10% FCS, without antibiotic antimycotic solution for toxin treatment, inhibitor treatment, and signal transduction experiments.5 THP-1 cells were incubated with CyaA  or ET  for different time durations in 200 µl of DMEM with 10% FCS and no antibiotics at 37 °C. The reaction was stopped by lysing the pellet by addition of 0.2% Tween-20 in 50 mM HCl. The lysates were boiled for 10 min at 100 °C, neutralized by addition of 150 mM unbuffered imidazole, and cellular cAMP levels were measured using competitive ELISA, as described elsewhere [6 × 10lsewhere . cAMP co6 cells). To investigate the effects of toxin-provoked cAMP intoxication on opsonophagocytic signaling, 3 × 106 THP-1 cells were preincubated with CyaA  for 30 min or ET  for 6 h, respectively   at a fin3VO4, 10 mM NaF, 0.3% SDS, 1% Triton X 100, and EDTA-free protease inhibitor cocktail ). The proteins were separated using SDS-PAGE and transferred onto polyvinylidene difluoride membranes. The blots were probed with indicated primary antibodies, followed by incubation with horseradish peroxidase-conjugated secondary antibody and developed using SuperSignal West Femto maximum sensitivity substrate . The chemiluminescent signal was recorded using a G: Box Chemi gel documentation system . The immunoblots were analyzed using AIDA Image Analysis software , and an AIDA 2D Densitometry module was used to evaluate the intensity of the chemiluminescent signal. The background-subtracted density of the bands was measured for each individual band from immunoblots probed by phospho-specific antibodies and normalized by the corresponding density of bands from immunoblots probed by antibodies recognizing the total form of the signaling protein concerned. The values obtained from the positive controls (Buffer + SOZ or Adefovir + SOZ) were set at 100%.Cellular lysates were analyzed by immunoblotting as previously described . Briefly5 THP-1 cells were incubated with CyaA or ET as mentioned above, washed with HBSS buffer and treated with serum opsonized FITC-zymosan particles (10 µg of zymosan particles per 105 cells) for 30 min at 37 °C or 4 °C in HBSS buffer. Toxin and opsonized FITC-zymosan particles were simultaneously added to the cells in some experiments. THP-1 cells were preincubated with adefovir dipivoxil for 6 h prior to treatment with toxin and subsequent incubation with opsonized FITC-zymosan in some experiments. The reaction was stopped by addition of 3 volumes of chilled HBSS buffer, the cells were centrifuged and resuspended in 100 µl of chilled HBSS buffer. Live THP-1 cells were selected by performing the flow cytometric analysis in the presence of Hoechst 33258 (1 µg/ml), and the cells were further selected (and FITC-zymosan particles excluded) using gating on a bi-dimensional cytogram based on signals from forward scatter and side scatter. Data were analyzed using FlowJo software . The number of cells that incorporated fluorescent zymosan particles was determined on a fluorescence histogram. Phagocytic activity was expressed as phagocytic index (PhI) [2 × 10ex (PhI) , calcula6 THP-1 cells were seeded on cover slips (2 h) in DMEM with 10% FCS and no antibiotics. The cells were then treated at 37 °C with CyaA  for 30 min or ET  for 6 h to obtain similar overall levels of cAMP intoxication. The toxin treated or untreated cells were incubated with serum opsonized zymosan or unopsonized zymosan particles (100 µg of zymosan per 106 cells) for 30 min at 37 °C. The cells were washed twice with warm PBS (37 °C), fixed by incubation with 3.7% paraformaldehyde for 15 min, and permeabilized by 0.1% Triton in PBS. The cover slips were blocked with 5% BSA in PBS for 1 h and stained with TRITC conjugated phalloidin dissolved in 2% BSA (1:50) in the presence of 1 μg/ml of DAPI. Finally, the coverslips were washed three times with PBS for five minutes each, rinsed once with distilled water, and mounted in the inverted position on a microscopic slide in Vectashield mounting medium. Immunofluorescent images were obtained using an Olympus FV-1000 confocal microscope . Reconstructed z stack projections (average intensity) of the images were used for measuring the cell area. The cell periphery was traced using freehand selection tools in ImageJ, the area enclosed was measured using the measure function and plotted as relative cell area, where the area of Zymosan treated cells was set as 1.0 (negative control).To visualize the actin cytoskeleton and the cell morphology, 3 × 10p value less than 0.05 was considered statistically significant. All statistical analyses were performed with Prism (version 6) software . The axis was split in some graphs to facilitate the accurate representation of the trends.One-way analysis of variance (ANOVA) was used to perform statistical analysis followed by Tukey’s test or Dunnett’s test for post hoc analysis. A


Reactive oxygen species (ROS) promote carcinogenesis by inducing genetic mutations, activating oncogenes, and raising oxidative stress, which all influence cell proliferation, survival, and apoptosis. Cancer cells display redox imbalance due to increased ROS level compared to normal cells. This unique feature in cancer cells may, therefore, be exploited for targeted therapy. Over the past few decades, natural compounds have attracted attention as potential cancer therapies because of their ability to maintain cellular redox homeostasis with minimal toxicity. Preclinical studies show that bioactive dietary polyphenols exert antitumor effects by inducing ROS-mediated cytotoxicity in cancer cells. These bioactive compounds also regulate cell proliferation, survival, and apoptotic and antiapoptotic signalling pathways. In this review, we discuss (i) how ROS is generated and (ii) regulated and (iii) the cell signalling pathways affected by ROS. We also discuss (iv) the various dietary phytochemicals that have been implicated to have cancer therapeutic effects through their ROS-related functions. Reactive oxygen species (ROS) are highly reactive metabolic by-products that cause both deleterious and beneficial effects. Cellular ROS act as secondary messengers in signalling cascades that are critical for normal physiological functions such as differentiation and development , 2. Howe2O2), which is subsequently catalyzed by glutathione peroxidase (GPX) to generate H2O. Catalase (CAT) also converts H2O2 to water , mitochondria, and peroxisomes  Figure . Endogento water  13]. Ot. Ot2O2),to water .Besides these antioxidants, the transcription factor, nuclear factor erythroid 2- (NFE2-) related factor 2 (Nrf2), also contributes in controlling oxidative stress. Activation of Nrf2 requires inhibition of its negative regulator Keap1, which results in Nrf2 nuclear translocation . This le2O2-mediated oxidation of protein cysteine residues, triggering specific cellular events such as proliferation piperidine) is the most abundant natural alkaloid found in long pepper (Piper longum L.). Recently, it was determined to be a promising anticancer compound . PiperinμM [κB pathways [L and increases BAX/caspase-3-associated apoptosis [1-ATPase, inhibiting mitochondrial ATP synthesis [WAF1 and p27KIP1 and downregulating cyclins D1, D2, and E and Cdks 2, 4, and 6 [Resveratrol , a polyphenol that is found in grapes and berries, effectively prevents tumor initiation and progression by stimulating apoptosis at 10 to 100 μM  in prostμM  and neurμM . ResveraμM . Resverapathways  and STATpathways . Moreovepoptosis . Resveraynthesis , 148. It4, and 6 , 150.p-Coumaric acid (p-CoA), an isomer of cinnamic acid [Peanuts, tomatoes, and carrots are rich in mic acid . In colomic acid , 152. Inmic acid .2-methoxy-1,4-naphthoquinone (MNQ) and 8-hydroxy-2-methoxy-1,4-naphthoquinone (HMNQ) elicit ROS production and induce apoptosis via the JNK/p38 MAPK pathway [The naturally occurring quinone compounds have potent cytotoxicity against cancer cells. In lung adenocarcinoma cells,  pathway –156.Naringenin, a citrus flavonoid, triggers ROS-induced apoptosis and stimulates p38MAPK-mediated caspase activation [tivation , 158.Gallic acid , which is widely present in grapes and red wine, inhibits lung cancer cell growth by increasing ROS level and depleting GSH [2O2 and O2−, leading to mitochondria-dependent apoptosis [ting GSH . In prospoptosis . GA alsopoptosis  and JNK poptosis .in vivo study showed that curcumin promotes lung cancer [Poor bioavailability is a major obstacle for natural bioactive compounds, especially for Qu, curcumin, and resveratrol, which are associated with poor absorption and fast metabolism in the liver and intestine. Pharmacokinetic profile analysis of Qu revealed that about 93% of the compound is metabolised after oral administration (10 mg/kg) in male Sprague-Dawley rats . On the g cancer  and topig cancer , suggestin vivo biodistribution.As dietary phytochemicals lack mechanistic selectivity, these natural compounds display a variety of effects in different cancer cell types and thus the discrepancies in results among separate studies. Other possible reasons for divergent findings in different studies include changes or differences in (i) stability of the bioactive compounds in cell culture medium, for example, stability of Qu decreases at pH 7 or 8 ; (ii) reNatural phytochemicals have been associated with anticancer properties through their ability to modulate oxidative stress, cell cycle regulators, and proapoptotic, antiapoptotic, and survival signalling pathways. In preclinical and clinical trials, bioactive compounds show a promising and wide therapeutic window against various malignancies, including glioblastoma and breast, colon, and prostate cancers where phytochemical-induced cancer cell death was observed. However, certain attributes such as poor solubility and bioavailability of these bioactive compounds limit their clinical application. Thus, further studies are required to identify ways for effective biological delivery of these compounds in different cancer cell types. It is also critical that detailed studies are conducted in large cohorts to establish the pharmacokinetic profile of these compounds alone and in combination with other chemotherapeutic agents to determine dosage, tissue targets, and toxicity. Indeed, natural phytochemicals may serve as future therapy for specific types of cancer.


Acne is the eighth most frequent disease worldwide. Inflammatory response runs through all stages of acne. It is complicated and is involved in innate and adaptive immunity. This study aimed to explore the candidate genes and their relative signaling pathways in inflammatory acne using data mining analysis. Microarray data GSE6475 and GSE53795, including 18 acne lesion tissues and 18 matched normal skin tissues, were obtained. Differentially expressed genes (DEGs) were filtered and subjected to functional and pathway enrichment analyses. Protein–protein interaction (PPI) network and module analyses were also performed based on the DEGs. In this work, 154 common DEGs, including 145 upregulated and 9 downregulated, were obtained from two microarray profiles. Gene Ontology and pathway enrichment of DEGs were clustered using significant enrichment analysis. A PPI network containing 110 nodes/DEGs was constructed, and 31 hub genes were obtained. Four modules in the PPI network, which mainly participated in chemokine signaling pathway, cytokine–cytokine receptor interaction, and Fc gamma R-mediated phagocytosis, were extracted. In conclusion, aberrant DEGs and pathways involved in acne pathogenesis were identified using bioinformatic analysis. The DEGs included FPR2, ITGB2, CXCL8, C3AR1, CXCL1, FCER1G, LILRB2, PTPRC, SAA1, CCR2, ICAM1, and FPR1, and the pathways included chemokine signaling pathway, cytokine–cytokine receptor interaction, and Fc gamma R-mediated phagocytosis. This study could serve as a basis for further understanding the pathogenesis and potential therapeutic targets of inflammatory acne. Acne is a common inflammatory skin disease affecting the pilosebaceous unit. Acne is the eighth most frequent disease worldwide, with a prevalence rate of 94% as evaluated by the Global Burden of Disease Project . Acne ca Propionibacterium acnes (P. acnes), increased sebum production, infundibular hyperkeratinization of the pilosebaceous unit, and inflammation, are involved in the pathogenesis of acne. Inflammation plays an important role in the progression of acne lesions  were calculated. The Benjamini & Hochberg false discovery rate method was used as a correction factor for the adjusted P-value in GEO2R. The statistically significant DEGs were identified according to P<0.05 and [logFC] ≥ 1. The common DEGs in GSE6475 and GSE53795 were filtered by the software Functional Enrichment analysis tool (FunRich), which was downloaded from the online website http://www.funrich.org/.The online tool GEO2R (https://www.ncbi.nlm.nih.gov/geo/geo2r/) was used to analyze the DEGs between AL and NS in the microarray data of GSE6475 and GSE53795, respectively. The adjusted P-value<0.05 and enrichment gene count>2.GO was used to define gene functions in three aspects: molecular function (MF), cellular component (CC), and biological process (BP). DAVID is an online website that provides a comprehensive set of functional annotation tools to understand the biological meaning behind a large list of genes. In the present study, the functional enrichment analyses of the statistically significant DEGs, including GO analysis and KEGG pathway enrichment analysis, were conducted using DAVID, with the cut-off criterion ofThe common DEGs of GSE6475 and GSE53795 were analyzed using the online website STRING , with 0.700 (moderate confidence) as the minimum required interaction score. Then, the software Cytoscape was used to establish a PPI network. The Network Analyzer in Cytoscape was utilized to calculate node degree. As an App in Cytoscape, CytoHubba was used to identify the hub genes in the PPI network. MCODE was used to perform modular analysis, with the parameters set as follows: a Degree Cutoff=2, Node Score Cutoff=0.2, K-Core=2, and Max. Depth=100. Finally, pathway enrichment analyses of candidate genes in each cluster of PPI network were performed using DAVID (https://david.ncifcrf.gov/).http://www.ehbio.com/ImageGP/index.php/Home/Index/Volcanoplot.html).The microarray datasets GSE6475 and GSE53795 were obtained from the public database GEO. We detected 162 upregulated and 13 downregulated genes in GSE6475 and 476 upregulated and 385 downregulated genes in GSE53795, with a threshold of P value<0.05 and fold change (FC) [logFC] ≥ 1. A total of 145 consistently upregulated and 9 concurrently downregulated DEGs were extracted from these two microarray datasets . A totalThe consistently upregulated DEGs were clustered via the online website DAVID for the functional and KEGG pathway enrichment analyses of DEGs in Acne. The GO analysis of DEGs can be divided into three components: MF, CC, and BP. In terms of MF, the upregulated DEGs were mainly involved in calcium ion binding, serine-type endopeptidase activity, receptor activity, and cytokine activity . As far  FPR2, ITGB2, CXCL8, C3AR1, CXCL1, FCER1G, LILRB2, PTPRC, SAA1, CCR2, ICAM1, and FPR1, all of which belonged to the common upregulated DEGs  , module  P. acnes participates in the inflammation of acne by targeting Toll-like receptors (TLRs) [α is supposed to initiate microcomedos; on the other part, P. acnes promotes the production of IL-1α by activating the Nod-like receptor 3 inflammasome in monocytes [ P. acnes; IL-17 was detected in the biopsy of AL [β, beta-defensins 1 and 2, IL-10, TNF-α, CXCL-2, and matrix metalloproteinases-1 (MMP-1), MMP-3, and MMP-9, can also be detected [Multiple agents are involved in the processes of acne inflammation. In terms of innate immunity,s (TLRs) . IL-1α ionocytes , 10. Othonocytes . In the onocytes . Th17 lysy of AL . Other cdetected . In the Bioinformatic analysis of microarray and/or sequencing data is widely used to explore the aberrant gene expression, potential pathogenesis, and therapeutic targets of various diseases. In the present study, 154 common DEGs, including 145 upregulated and 9 downregulated, were identified in datasets GSE6475 and GSE53795 from GEO. The PPI network contained 31 hub genes, with the addition of the candidate genes in each module, both of them belonged to the upregulated DEGs. Considering the minor number and roles of the downregulated DEGs, we focused on the aberrant upregulated DEGs and their relative molecular mechanisms in acne. SAA1, CCR2, C3AR1, FPR2, FPR1, CXCL1, and CXCL8 were listed in the top 12 hub genes, and these genes mainly participated in chemokine signaling pathway, cytokine–cytokine receptor interaction, and NOD-like receptor signaling pathway. SAA1, namely, serum amyloid A-1 protein which belongs to the SAA family, is a major and highly conserved acute phase protein. SAA1 and SAA2 (another member of SAA family) contribute to inflammatory skin diseases such as acne. Su Q et al. showed that glucocorticoids in combination with P. acnes increase SAA1 and SAA2 expression, whereas glucocorticoids promote SAA1 production by combining with TNF, indicating that glucocorticoids induce SAA1 expression under infectious and sterile inflammatory circumstances, promoting the progress of cutaneous inflammation [β, IL-8, IL-12, and RANTES, positively correlate with acne under mustard gas-exposed conditions [Using the app CytoHubba in Cytoscape, we filtered 31 hub genes in the PPI network. Module 1 contained 14 genes, all of which belonged to hub genes. Among these genes,ammation . CCR2  is a lipid kinase containing a regulatory and a catalytic subunit. PIK3R2 is a regulatory subunit of PI3K, which phosphorylates phosphatidylinositol 4,5-bisphosphate to produce phosphatidylinositol 3,4,5-trisphosphate and recruits PH domain-containing proteins, such as Akt1, to the membrane. Activation of the PI3K-Akt pathway plays a role in cell survival, proliferation, and motility [α, which is associated with acne development [Genes of Module 3 includedmotility . Shi G emotility . Suppreselopment . Activatelopment . SERPINA1, VCAN, IGFBP4, SPP1, TNC, and TIMP1, in which TNC and SPP1 were clustered and participated in ECM–receptor interaction. The extracellular matrix (ECM) consists of a variety of macromolecules. The combination of ECM and specific transmembrane molecules (mainly integrins) of cells plays a role in cellular activities, including proliferation, apoptosis, differentiation, adhesion, and migration. Both TNC and SPP1 are matricellular proteins that are upregulated in active tuberculosis (TB) relative to healthy controls or latent TB; OPN  and TNC may function as reliable biomarkers for monitoring TB activity [α4 integrin and PI3K/Akt pathway [Genes of Module 4 includedactivity . TNC facactivity . Both TNactivity . OPN is  pathway . Tear OP pathway . However FPR2, ITGB2, CXCL8, C3AR1, CXCL1, FCER1G, LILRB2, PTPRC, SAA1, CCR2, ICAM1, and FPR1, and the possible important pathways are chemokine signaling pathway, cytokine–cytokine receptor interaction, and Fc gamma R-mediated phagocytosis. These results could help elucidate the molecular mechanism underlying acne pathogenesis and provide potential targets for acne therapy. Further investigations are needed to confirm our putative finding.In conclusion, the present study exhibits the global profile of DEGs and relative signaling pathways that might participate in the initiation and development of acne mechanically. In the pathogenesis of acne, the possible crucial genes are


Mononuclear cell infiltration of exocrine glands, production of Ro/SSA and La/SSB autoantibodies, along with oral and ocular dryness, are characteristic features of primary Sjögren’s syndrome (pSS). Non-SS sicca subjects, an underexplored group in relation to pSS, display similar sicca symptoms, with possible mild signs of inflammation in their salivary glands, yet with no serological detection of autoantibody production. In this study, we investigated inflammatory manifestations in the salivary gland tissue, tear fluid and saliva of non-SS subjects, as compared to pSS patients and healthy individuals.Fifteen non-SS, 10 pSS and 10 healthy subjects were included in the analyses. Histological evaluation of salivary gland biopsies was performed. Liquid chromatography-mass spectrometry (LC-MS) was conducted on tear fluid and stimulated whole saliva, and proteomic biomarker profiles were generated. Extracellular vesicle (EVs) isolation and characterisation from both fluids were also combined with LC-MS. The LC-MS data were analysed for quantitative differences between patient and control groups using Scaffold. Database for Annotation, Visualization and Integrated Discovery (DAVID) and Functional Enrichment Analysis Tool (FunRich) were applied for functional analyses.Histopathological evaluation of salivary gland biopsies showed implications of milder inflammation in non-SS subjects through mononuclear cell infiltration, fibrosis and fatty replacement, as compared to pSS patients. Although unaffected in the non-SS group, upregulation of proinflammatory pathways and proteins involved in ubiquitination (LMO7 and HUWE1) and B cell differentiation (TPD52) were detected in tear fluid of pSS patients. Moreover, overexpression of proteins STOM, ANXA4 and ANXA1, regulating cellular innate and adaptive immunological pathways, were further identified in EVs from tear fluid of pSS patients. Finally, whole saliva and EVs isolated from whole saliva of pSS patients expressed proteins vital for innate MHC class I cellular regulation (NGAL) and T cell activation (CD44).Non-SS sicca subjects may show implications of mild inflammation in their glandular tissue, while their protein profile was strikingly more similar to healthy controls than to pSS patients. Hence, the tear and salivary biomarkers identified could be implemented as potential non-invasive diagnostic tools that may aid in increasing diagnostic accuracy when evaluating non-SS subjects and pSS patients and monitoring disease progression.The online version of this article (10.1186/s13075-019-1961-4) contains supplementary material, which is available to authorized users. In some cases, subjects may display sicca symptoms and may show some mild infiltration of mononuclear cells in their exocrine glands, yet serologically no autoantibody production is detected [Primary Sjögren’s syndrome (pSS) is a systemic rheumatic autoimmune disease that is characterised by chronic inflammation, autoantibody production and destruction of exocrine glands through mononuclear cell infiltration. The primary target organs are the lacrimal and salivary glands , 2, resudetected . Hence, Interestingly, the destruction of salivary gland tissue through the deregulated infiltration and proliferation of lymphocytes may also lead to the formation of ectopic germinal centre (GC)-like structures in approximately 20% of pSS patients –14. It iIn view of currently available diagnostic tools for pSS, there is an unmet need for the incorporation of non-invasive, more accurate diagnostics. Studying the proteome of biological fluids and screening for disease-specific biomarkers  through Various separation techniques can also be coupled with proteomic analyses, in order to isolate cellular components of interest when screening for disease biomarkers. Extracellular vesicles (EVs) are an example of such cellular components, comprising of exosomes (size < 100 nm), microvesicles (size 100–1000 nm), and apoptotic bodies (size 1000–5000 nm) . InteresWe have previously applied LC-MS using samples of stimulated whole saliva and tear fluid from patients with pSS and healthy controls, in combination with EV isolation, which resulted in the detection of potential novel disease biomarkers , 44. To Fifteen non-SS sicca subjects, 10 pSS patients that fulfilled the AECG classification criteria from 2002  and 10 a.Routine haematoxylin and eosin-stained sections from minor salivary gland biopsies of the non-SS and pSS subjects included in the study were evaluated using a light microscope . Both mononuclear cells in focal infiltrates and those located interstitially, i.e., in close proximity to the acinar or ductal epithelium, were analysed. Additionally, other forms of tissue damage, including fibrosis, in the same area were also investigated. Furthermore, these salivary gland sections were scored blindly for the presence of fatty infiltration, as previously described , 17. DepParticipants underwent a thorough ocular surface examination at the Norwegian Dry Eye Clinic, and a detailed oral examination at the Dry Mouth Clinic, where tear fluid and stimulated whole saliva were collected, respectively, as previously described , 45. In EVs were isolated from tear fluid and stimulated whole saliva using size-exclusion chromatography, as described previously . In brieIn order to characterise the isolated EVs, nanoparticle tracking analysis and immunoaffinity capture for detection of CD9 positive EVs were conducted on joint fractions from saliva and tears as previously described . In brieg for 20 min at 4 °C , and then re-dissolved in 50 μl of a mixture of 6 M urea and 100 mM ammonium bicarbonate (pH 7.8), followed by reduction and alkylation of cysteines. The alkylation reaction was quenched, and the proteins were digested with 10 μg of trypsin for 16 h at 37 °C to generated peptides that were then purified using an OMIX C18-micro SPE  and dried using a Speed Vac concentrator . These tryptic peptides were analysed using an Ultimate 3000 RSLCnano-UHPLC system connected to a Q Exactive mass spectrometer  and a nano electrospray ion source.Proteomics analysis was performed on saliva and tears from non-SS sicca subjects, pSS patients, and healthy controls before and after isolation of EVs, as previously described . In brieFor liquid chromatography separation, an Acclaim PepMap 100 column   was used. A flow rate of 300 nL/min was employed with a solvent gradient of 4–35% B in 60 min. Solvent A was 0.1% formic acid, and solvent B was 0.1% formic acid/90% acetonitrile.R = 70,000 at m/z 200, after accumulation to a target of 1e6. The maximum allowed ion accumulation times were 60 ms. The method used allowed sequential isolation of up to the ten most intense ions, depending on signal intensity (intensity threshold 1.7e4), for fragmentation using higher-energy collisional induced dissociation (HCD) at a target value of 1e5 charges, NCE 28, and a resolution R = 17,500. Target ions already selected for MS/MS were dynamically excluded for 30 s. The isolation window was m/z = 2 without offset. For accurate mass measurements, the lock mass option was enabled in MS mode.The mass spectrometer was operated in the data-dependent mode to automatically switch between MS and MS/MS acquisition. Survey full-scan MS spectra (from m/z 400 to 2000) were acquired with the resolution Finally, data were acquired using Xcalibur v2.5.5, and raw files were processed to generate peak lists in Mascot generic format (*.mgf) using ProteoWizard release version 3.0.331. Database searches were performed using Mascot in-house version 2.4.0 to search the SwissProt database , as before .p < 0.05). For functional analysis of the proteomics data, Database for Annotation, Visualization and Integrated Discovery (DAVID)  and Functional Enrichment Analysis Tool (FunRich) (http://www.funrich.org/) were applied. Tear fluid, stimulated whole saliva, and EVs (joint fractions) were analysed individually, comparing pSS patients with the non-SS sicca subjects and the healthy controls, correspondingly. DAVID was applied, using a False Discovery Rate (FDR) with a maximum 5% cut-off, in order to delineate specific cellular pathways involving these upregulated proteins in the pSS patients, while FunRich was used to visualise the percentage of proteins involved in each of these upregulated signalling pathways.In order to validate MS/MS-based peptide and protein identifications, Scaffold  was used, as before . Here, pIn order to account for the morphological patterns of chronic inflammation in minor salivary gland biopsies of the non-SS and pSS subjects included in the study, the sections were evaluated for mononuclear cell infiltration, other form of tissue damage including fibrosis, and for the presence of fatty infiltration , 17. Intp values < 0.05 that were distinguished using spectral counts , E3 ubiquitin-protein ligase HUWE1 (HUWE1) and Tumour protein D52 (TPD52), in descending order. The most upregulated protein in tear fluid of patients with pSS, namely LMO7, is involved in ubiquitination and cell adhesion. Moreover, HUWE1, also mediates ubiquitination, in addition to neutrophil degranulation and cell differentiation. Lastly, TPD52 plays a central role in regulating B cell differentiation , metabolic processes, regulation of NF-kappaB import, erythrocyte homeostasis, MAP kinase cascade, removal of superoxide radicals, regulation of programmed cell death, natural killer cell cytotoxicity and activation, response to oxidative stress, as well as regulation of cell proliferation and apoptotic processes, in downward order, as determined by the percent values of the upregulated proteins contributing to each cellular process , Annexin A4 (ANXA4) and Annexin A11 (ANXA11). STOM is involved in neutrophil degranulation and regulation of viral genome replication, while ANXA4 affects NF-kappaB binding, apoptosis, and interleukin-8 secretion. Finally, ANXA11 regulates MHC class II protein complex binding and phagocytosis.The LC-MS conducted on stimulated whole saliva from 15 non-SS sicca subjects, 10 pSS patients and 10 healthy controls aided in the identification of significantly upregulated proteins in the patient group when compared to non-SS sicca subjects and healthy controls, respectively , CD44 antigen (CD44) and B2MG. The most upregulated of these proteins, FKBP1A, plays a role in T cell activation in FOXP3 expression and regulatory T cell suppression, in addition to promoting tumour growth and progression. Meanwhile, B2MG mediates antigen processing and presentation on MHC class I, and innate immunity. When comparing patients with pSS to healthy controls, proteins Secreted Ly-6/uPAR-related protein 1 (SLUR1), B2MG, and Clusterin (CLUS) were highly expressed in the patient group, in declining order. SLUR1 affects acetylcholine receptor activity, cell migration and proliferation, while CLUS plays modulates NF-kappa-B activity and TNF production , and Neutrophil gelatinase-associated lipocalin (NGAL), also referred to as LCN2. MVP promotes IFNγ-mediated signalling, MAP kinase activity and neutrophil degranulation, while NGAL is a tumour-associated antigen involved in cell adhesion and innate immunity. Comparing the pSS patient group with healthy controls helped distinguish proteins Ficolin-1 (FCN1), CD44 and ANXA4 as upregulated in the patient group, in descending order. The most changed of these proteins in EVs from whole saliva, FCN1, is a pattern-recognition receptor involved in innate immunity and complement activation  pathway overrepresentation analyses were performed using DAVID. Our results demonstrated pathways in the pSS patients involving inflammatory innate immune responses, such as MHC class I antigen processing and presentation, TNF-mediated signalling and IL-1 mediated signalling. Additional T cell receptor signalling of the adaptive immune response, and apoptotic processes through MAP kinase cascade, were also detected Fig. . InteresOur proteomic analysis of EVs extracted from pooled tear samples revealed upregulated cellular pathways in pSS patients, as compared to non-SS sicca subjects, involving retina homeostasis, regulation of metabolic processes, programmed cell death, natural killer cell cytotoxicity and cell proliferation. Correspondingly, the most upregulated of cellular processes in the patient group when compared to the healthy controls was also retina homeostasis, in addition to other adaptive and innate immune responses Fig. . Still, The DAVID analysis performed on highly expressed proteins identified in whole saliva, and EVs separated from whole saliva, did not disclose significantly affected signalling pathways in the pSS patient group. Nevertheless, when considering the number of spectral counts, we managed to identify the three most overexpressed proteins in the whole saliva of pSS patients  , and p < 0.001 is highlighted by (**). (TIFF 10515 kb)Figure S1. Upregulated protein expression of potential disease biomarkers identified in tear fluid of pSS patients. Considering the mean number of spectral counts for the proteins detected in the study groups included in our LC-MS analyses, the three most upregulated proteins in pSS patients (black) that are involved in immunological reactions, when compared to both non-SS subjects (grey) and healthy controls (white), were LMO7, HUWE1, and TPD52, in descending order. Statistical significance where Additional file 2:p < 0.01 is indicated by (*), and p < 0.001 is highlighted by (**). (TIFF 11015 kb)Figure S2. Upregulation of potential disease biomarkers detected in stimulated whole saliva of pSS patients. In view of the mean number of spectral counts of the proteins identified when performing LC-MS analyses, the three most overexpressed proteins in the pSS patient group (black) when compared to the non-SS sicca participants (grey) were FKBP1A, CD44, and B2MG. Meanwhile, when comparing patients with pSS (black) to healthy controls (white), proteins SLUR1, B2MG, and CLUS were highly expressed in the patient group, in declining order. Statistical significance where Additional file 3:p < 0.01 is indicated by (*). (TIFF 10937 kb)Figure S3. Overexpression of proteins and potential disease biomarkers found in EVs isolated from stimulated whole saliva in pSS patients. Viewing the mean spectral counts of proteins identified in EVs of whole saliva, the three most upregulated proteins in patients with pSS (black), as related to non-SS sicca participants (grey), included CD44, MVP, and NGAL, also referred to as LCN2. Comparing the pSS patient group (black) with healthy controls (white) helped distinguish proteins FCN1, CD44 and ANXA4 as upregulated in the patient group, in decreasing order. Statistical significance where Additional file 4:Table S1. Upregulated proteins in tear fluid of non-SS subjects vs. pSS patients. (PDF 277 kb)Additional file 5:Table S2. Upregulated proteins in tear fluid of controls vs. pSS patients. (PDF 262 kb)Additional file 6:Table S3. Upregulated proteins in whole saliva of non-SS subjects vs. pSS patients. (PDF 177 kb)Additional file 7:Table S4. Upregulated proteins in whole saliva of controls vs. pSS patients. (PDF 40 kb)Additional file 8:Table S5. Upregulated proteins in EVs isolated from whole saliva of non-SS subjects vs. pSS patients. (PDF 206 kb)Additional file 9:Table S6. Upregulated proteins in EVs isolated from whole saliva of controls vs. pSS patients. (PDF 211 kb)


Caffeine is commonly taken via the daily dietary consumption of caffeine-containing foods. The absorbed caffeine is metabolized to yield various metabolites by drug-metabolizing enzymes, and measuring the levels of each caffeine metabolite can provide useful information for evaluating the phenotypes of those enzymes. In this study, the urinary concentrations of caffeine and its 13 metabolites were determined, and the phenotypes of drug metabolic enzymes were investigated based on the caffeine metabolite ratios. Human urine samples were pretreated using solid phase extraction, and caffeine and its metabolites were analyzed using liquid chromatography-tandem mass spectrometry. Based on the urinary caffeine metabolite concentrations, the caffeine metabolite ratios were calculated for six human subjects at specified time points after caffeine intake. Variations in urinary metabolite levels among individuals and time points were reported. In addition, the resultant enzyme activities showed different patterns, depending on the metabolite ratio equations applied. However, some data presented a constant metabolite ratio range, irrespective of time points, even at pre-dose. This suggests the possibility of urinary caffeine metabolite analysis for routine clinical examination. These findings show that urinary caffeine and the metabolite analysis would be useful in evaluating metabolic phenotypes for personalized medicine. Caffeine, an alkaloid of the methylxanthine class, is the world’s most widely consumed psychoactive substance. As a naturally occurring substance, caffeine is found in the leaves, fruits, or seeds of more than 60 plant species. Caffeine is popularly and extensively taken via the daily dietary consumption of caffeine-containing beverages or foods ,2.In the liver, caffeine is subjected to a series of metabolic reactions to yield a mixture of N-methylated xanthines, uric acids, and an acetylated uracil, as its metabolites . There aSeveral analytical methods have been reported for measuring caffeine and its metabolites in urine using high-performance liquid chromatography (HPLC) or high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) ,14,15,16In this study, the urinary concentrations of caffeine and its metabolites were determined using LC-MS/MS analysis. The resulting concentration data was applied to various caffeine metabolite ratio equations to determine the phenotypes of each drug metabolic enzyme. The feasibility of phenotyping the drug-metabolizing enzyme based on urinary caffeine metabolite ratios was examined.Chemicals including, 1-methylxanthine (1X), 3-methylxanthine (3X), 7-methylxanthine (7X), 1,3-dimethylxanthine , 1,7-dimethylxanthine , 3,7-dimethylxanthine , 1,3,7-trimethylxanthine , 1-methyluric acid (1U), 1,3-dimethyluric acid (13U), 1,7-dimethyluric acid (17U), 3,7-dimethyluric acid (37U), and 1,3,7-trimethyluric acid (137U), and acetic acid were provided by Sigma-Aldrich . The following chemicals were procured from Santa Cruz : 5-acetylamino-6-amino-3-methyluracil (AAMU), 5-acetylamino-6-formylamino-3-methyluracil (AFMU) and internal standards (IS) including 1-methylxanthine-2,4,5,6-13C4 (1X*), 1,3,9-15N3, and 1-methyluricacid-2,4,5,6-13C4,1,3,9-15N3 (1U*). HPLC-grade acetonitrile was purchased from J. T. Baker . Water was prepared using a Milli-Q purification system . All other chemicals used were of analytical grade and used as received. All the standard solutions and mobile phases were passed through a 0.22-µm membrane filter before use.The study protocol and consent forms were approved by the Institutional Review Board of the Hanyang University, and all the participants provided written informed consent to participate in the study. The eligibility criteria for the study included physically healthy ethnic Korean adult men (19 years of age or older) who signed written informed consent. Participants were excluded if they were being treated for acute disease or other diseases or who needed treatment, or were receiving any medication that might affect the metabolism or excretion of caffeine. Urine samples were collected from 6 volunteers prior to the consumption of a caffeine-containing drink (120 mg of caffeine intake), and 1 h, 2 h, 4 h, 6 h, 8 h, and 10 h after the drink. Blank urine samples were obtained from healthy volunteers who had not consumed any methyl xanthine-containing food or beverage for the last 24 h. The urine samples were collected in clear 15-mL centrifuge tubes. All the study procedures were conducted in compliance with the principles of Declaration of Helsinki and Korean Good Clinical Practice guidelines (IRB HYG-16-193-2).For the LC–MS/MS analysis, 100 µL of urine was added to 10 mL of 0.1% acetic acid with IS. Then, 1 mL of diluted mixture was passed through pre-activated Sep-Pak C18 cartridges . The cartridge was washed with 1 mL of 0.1% formic acid two times, and then eluted with 1 mL of methanol. The eluate was dried under nitrogen gas. The residue was resolved in 0.1% acetic acid/acetonitrile , and a 5-µL aliquot was injected into the HPLC column for LC-MS/MS analysis. The analyte mixture was dissolved in MeOH at a concentration of 1 mg/mL and diluted to a series of working standard solutions. A 5-µL aliquot of each working standard solution was spiked to 95 µL of human blank urine. Then, the spiked samples were pretreated as described above. The concentrations of QC samples for each analyte are provided as The developed method was validated according to the US Food and Drug Administration (FDA) guidelines as mentioned in the “Guidance for Industry, Bioanalytical Method Validation, 2018” .The selectivity of the method was assessed by comparing multiple reaction monitoring (MRM) chromatograms between a blank sample and a standard spiked mixture. Lower limits of quantitation (LLOQs) for each analyte were determined considering the concentration level found in human urine samples, and evaluated for accuracy and precision. The calibration curves were prepared using the samples at concentration ranges depending on their LLOQ. The calibration curves were generated by plotting the peak area ratios of the analytes/IS versus the concentrations in the standard spiked samples. The linear correlation coefficient (r2) for all the calibration curves should be greater than 0.99.n = 3) on the same day. In case of inter-day assays, the precision and accuracy were assessed by determining the QC samples over three consecutive days. The accuracy was measured as a deviation of the calculated mean value from the nominal mean value, which should be within 15% of the nominal value except for LLOQ, which should not exceed 20% of the nominal value. The precision was determined at each concentration level, in terms of percent relative standard deviation (%RSD), which should not exceed 15% of the nominal concentration, except for the LLOQ, where it should not deviate by more than 20%.To assess the intra-day precision and accuracy, QC samples were analyzed, in triplicate, at different concentration levels  and TSQ Quantum™ Access MAX Triple Quadrupole Mass Spectrometer , equipped with an electrospray ionization (ESI) source. Chromatographic separation was achieved on a Kinetex C18 column  at a temperature of 40 °C. The HPLC mobile phases consisted of two solvents: (A) 0.1% acetic acid and (B) acetonitrile in 0.1% acetic acid. A linear gradient program was used with a flow rate of 0.2 mL/min. The initial mobile phase was set at 15% of solvent B and gradually increased to 90% in 3 min, kept at 90% for 1 min, and then followed by re-equilibrium for 3 min. Electrospray ionization (ESI) was performed in both positive and negative ion mode, with nitrogen as the nebulizing agent, spray voltage, sheath gas pressure, and aux gas pressure at optimal values of 3000, 60, and 20 (arbitrary units), respectively. The capillary temperature was 350 °C. Multiple reaction monitoring (MRM) detection was employed. The precursor–product ion pairs used in MRM mode are provided as The urinary caffeine and its metabolite concentrations were measured using the LC-MS/MS analysis. The resulting data were evaluated using the equations for the metabolic ratio calculation. The molar urinary ratios specific for each drug-metabolizing enzyme were calculated referring to the equations previously reported ,17,18,20+) at m/z 195.2, 167.1, 167.0, 181.2, 181.2, 197.2, 197.1, 197.1, 211.2, 199.2, and 174.1, respectively. Additionally, 3X, 13X, 1U, AFMU, and 1U* were ionized to yield the deprotonated molecular ions ([M−H]−) at m/z 164.9, 179.1, 181.1, 225.1, and 188.1, respectively. Ion polarity switching was applied for the simultaneous detection of protonated and deprotonated ions. Water and acetonitrile were used as the mobile phase solutions; to increase the response of 1U, 0.1% acetic acid was added to both mobile phase solvents. Using the gradient elution, all the analytes were eluted within 5 min. The representative LC-MS/MS extracted ion chromatograms are provided in the Caffeine, 1X, 7X, 17X, 37X, 13U, 17U, 37U, 137U, AAMU, and 1X* were ionized to yield the protonated molecular ions  values greater than 0.99. The linear ranges for caffeine and its metabolites are presented in the The intra-day precision was less than 16.4%, while the accuracy  was within the range of ±11.9% at the tested QC concentrations. The inter-day assay also showed satisfactory accuracy and reproducibility, with a precision of less than 11.4%, and an accuracy within the range of ±14.0% at the tested QC levels. These results are summarized in The matrix effect was negligible for caffeine and its metabolites, except for 1X, 137U, and AAMU, which seemed to be affected by it. However, they showed acceptable RSD criteria (within ±15%). For recovery evaluation, caffeine and all its metabolites were stable and well recovered  from samples. The matrix effect and recovery data are provided in the In all the tested conditions, caffeine and its metabolites were shown to be stable, with acceptable recovery (RSD within ±15%), except for the long-term stability of AAMU and AFMU . The accThe urinary caffeine metabolite levels in the six subjects are as shown in Subsequently, enzyme-specific metabolite ratios were calculated based on the urinary concentration data. Referring to the extant literature, the urinary metabolite concentrations were applied to various equations to yield enzyme-specific metabolite ratios . Figure To investigate the CYP1A2 phenotypes, seven equations were tested. The resulting metabolic ratio plots are shown in To investigate the CYP2A6 phenotypes, two equations were tested . Figure NAT2 activity was tested with four different equations. Three equations a–c generThe XO activity was evaluated using two equations. The metabolite ratio plots are shown in This study measured the urinary concentrations of caffeine and its 13 metabolites for 8 h, including a pre-dose time point, using LC-MS/MS. The metabolic ratios for phenotyping the drug metabolizing enzymes were calculated based on the various equations previously reported. Subsequently, the resulting metabolic ratios or patterns were compared, and their validity and feasibility were investigated.The resulting urinary caffeine concentration data showed a relatively obvious increase and decrease pattern across time, which indicated the absorption and elimination of caffeine after oral intake. Meanwhile, the changes in caffeine metabolite concentration levels, according to time points, were not as evident as those of caffeine. However, caffeine metabolites also showed a weak pattern of slow increase after caffeine intake, on excluding the data at pre-dose. Most metabolites showed the highest concentrations at pre-dose, which was presumably due to the urine concentration. Thus, the urine was diluted in post-dose samples, as the urine was frequently collected  after caffeine intake; meanwhile, at pre-dose, the caffeine metabolites, which resulted from usual dietary caffeine intake, could be detected at higher concentrations in relatively concentrated urine.The most diverse equations have been suggested to determine the enzyme activity of CYP1A2 in previous research. This study applied seven equations to yield the metabolite ratio. Among them, three equations a–c showeAcetylation is a primary route for the biotransformation of many hydrazine drugs, which is mediated by NAT . The polMeanwhile, the time point for sample collection may affect the metabolic ratio results, as the rate of metabolic reaction may be different depending on each metabolic pathway. Jetter et al. 2009) reported that the timing of urine collection can affect XO phenotyping results [09 reportRecently, research on polymorphism and personalized medicine has been extensive. Genotyping and phenotyping for drug metabolizing enzymes are vital strategies for characterizing the polymorphism of drug-metabolizing enzymes in individuals for personalized medicine. However, evaluating the phenotype is more critical than evaluating the genotype in some enzymes. For example, XO is a form of xanthine oxidoreductase, which is a type of enzyme that generates reactive oxygen species . These eMeanwhile, it has been recognized that differences in the activity of enzymes involved in nicotine metabolism are partly responsible for inter-individual variation in lung cancer risk among smokers ,33,34,35The limitations of this study are its small sample size, the lack of genotyping data, and that this data was obtained from a single set of experiments. Nevertheless, this study enables the evaluation of phenotyping results by demonstrating the caffeine metabolite ratio plots generated from different phenotyping equations. In addition, the data obtained at different time points, including the pre-dose and post-dose suggests the possibility that the enzyme phenotyping for CYP1A2, CYP2A6, NAT2, and XO can be conducted as a routine urine test, without the administration of drugs.Caffeine is popularly present in a wide variety of foods and beverages, and is extensively consumed via the daily diet. By measuring the exposure to caffeine in biological samples, such as the urine, the extent of caffeine consumption could be directly indicated. However, caffeine can be also used as a biomarker to indicate the activities of drug-metabolizing enzymes. This study demonstrated the possibility that enzyme phenotyping based on urinary caffeine metabolite ratios can be routinely used under general dietary conditions. This suggests a possibility for urinary caffeine metabolite analysis as a routine clinical examination. Urinary caffeine and its metabolite analysis would be useful in evaluating drug metabolic phenotypes for personalized medicine.