Coffee ingestion significantly increased salivary caffeine levels  and CON  trials, with men and women both improving by approximately 9 seconds and 6 seconds following coffee ingestion compared with placebo and control, respectively. However, no differences were observed between CON and PLA . In conclusion, ingesting coffee providing 3 mg·kg-1 of caffeine increased salivary caffeine levels and improved 5 km cycling time trial performance in men and women by a similar magnitude.Caffeine is a well-established ergogenic aid, although research to date has predominantly focused on anhydrous caffeine, and in men. The primary aim of the present study was to investigate the effect of coffee ingestion on 5 km cycling time trial performance, and to establish whether sex differences exist. A total of 38 participants (19 men and 19 women) completed a 5 km time trial following the ingestion of 0.09 g·kg Consequ, coffee , and in , coffee .Studies examining caffeine and exercise performance in women have produced varied results, with some reporting an ergogenic effect , whilst -1 of caffeine may be used as an alternative to anhydrous caffeine to improve endurance performance. Furthermore, recent reviews suggest that coffee ingestion should exert an ergogenic effect in most individuals [There is growing evidence, albeit predominantly in men, that similar ergogenic benefits can be obtained from caffeine and coffee ingestion. Graham et al.  concludeividuals  with a sividuals . Therefo-1·min-1, body fat percentage: 26 ± 8%; physical activity  [–1; 19 women: age: 28 ± 6 years, height: 166 ± 7 cm, body mass: 72 ± 11 kg, -1·min-1 body fat percentage: 33 ± 6%; physical activity (IPAQ [–1) completed a 5 km cycling time trial on a cycle ergometer following the ingestion of 0.09 g·kg-1 coffee (COF), a placebo (PLA) or control . Men had a higher body mass (t(36) = 2.621; p = 0.013; 95%CI: 2, 17; d = 0.86), fat-free mass (t(36) = 8.769; p < 0.001; 95%CI: 14, 22; d = 2.91) and t(36)=2.985; p = 0.005; 95%CI: 3, 15; d = 0.97) but lower fat mass (t(36) = −8.631; p < 0.001; 95%CI: −17, −10; d = 2.81) and percentage body fat (t(36) = −4.156; p < 0.001; 95%CI: −12, −4; d = 1.36). There was no significant difference in physical activity (t(36) = 0.629; p = 0.533; 95%CI: −1042, 1981; d = 0.20), and hence, both men and women were defined as highly active [In a double-blind, Latin-square randomised, crossover, placebo-controlled design, 38 recreationally active participants (19 men: (Mean ± SD) age: 30 ± 5 years, height: 179 ± 6 cm, body mass: 81 ± 12 kg, maximal oxygen uptake (e (IPAQ) ): (mediaty (IPAQ ):  and a familiarisation of the 5 km time trial, performed on the same day. On arrival at the laboratory, nude body mass  and body composition  were measured after voiding. Participants adjusted the saddle and handlebars for comfort, and these adjustments remained consistent for all trials. After a 10 min warm-up, the test protocol began at 100 W and increased by 25 W every 2 min until volitional exhaustion. The warm-up consisted of a two-minute cycle at 70–80 rpm with air resistance set at “level 1” followed by an 8 min progressive build up to each participant’s peak power output achieved during the graded exercise test. The protocol consisted of 2 min of cycling at a self-selected power output, a further 2 min of self-paced cycling, including three 6 s maximal sprints and finishing with 3 min of cycling at a self-selected power output . Breath--1 and >300 mg·day-1 defined as high [−1; Women: mean ± SD: 214 ± 158 mg·day−1 and range: 0–576 mg·day−1), although there were no significant differences between sexes (t(36) = −0.524; p = 0.604; 95%CI: −116, 68; d = 0.16). In addition, a 24 h dietary record was completed by each participant prior to the first trial; it was then analysed , photocopied and returned to the participants, so that the same diet could be repeated for subsequent trials; daily energy: Men: 2160 ± 634 kcal vs. Women: 2043 ± 591 kcal (t(36) = 0.587; p = 0.561; 95%CI: −287, 520; d = 0.19); Carbohydrate: Men: 229 ± 87 g vs. Women: 222 ± 99 g (t(36) = 0.217; p = 0.829; 95%CI: −55, 68; d = 0.08); Protein: Men: 111 ± 45 g vs. Women: : 96 ± 27 g (t(36) = 1.211; p = 0.234; 95%CI: −10, 39; d = 0.40); Fat: Men: 83 ± 35 g vs. Women: 81 ± 31 g (t(36) = 0.129; p = 0.898; 95%CI: −20, 23; d = 0.06). Participants were also instructed to abstain from caffeine, alcohol and strenuous activity for at least 12 h, which along with diet was confirmed verbally prior to each trial. An overnight caffeine abstinence was employed as Pickering and Kiely [−1 dose of caffeine has been reported to significantly improve exercise performance irrespective of whether a 4 days withdrawal period was imposed on habitual caffeine users [Habitual caffeine consumption was assessed using an adapted version of the Buhler et al.  caffeine as high   was used in the coffee trials and dissolved in 300 mL of hot water (58 ± 3°C) and served in lidded cups. Participants were given a maximum of 10 min to consume the beverage, and the 60 min rest period started once the cup was emptied . The plaFollowing the rest period, participants performed the same ten min warm-up as described earlier. Following completion of the warm-up, there was a 60 s period where the participants were instructed to sit passively before a standardised countdown to initiate the time trial. The participants then performed a 5 km cycling time trial where they were instructed to complete the distance as fast as possible. During each time trial, participants had access to the distance remaining, and with the exception of verbal encouragement, no other information was provided. The gearing was self-selected by the participants on the Wattbike during the familiarisation trial and then replicated during each time trial. Bellinger and Minahan  reportedHeart rate (HR) was measured throughout each time trial using a short-range telemetry HR transmitter strap . Saliva samples (minimum 0.5 mL by the passive drool technique) were obtained immediately before fluid ingestion to establish compliance with the washout period, one-hour post-ingestion prior to commencing the time trial and 60 s following the time trial. Participants were instructed to expectorate into a 20 mL plastic cup, and the sample was then transferred to a capped glass vial that was immediately placed in a −80°C freezer for subsequent analysis of caffeine concentration using a standard enzyme-linked immunoassay kit . At the same time points, a capillary blood sample was drawn from the index finger for determination of blood glucose and lactate concentrations . Feeling scale (FS) , felt arA statistical power analysis was performed for sample size estimation based on the race time from a previous coffee-ingestion study . As thisby Cohen , were al2,61=10.761; p < 0.001; -1 of caffeine. Performance in the coffee trial (482 ± 51 s) was faster than the placebo  and control  trials. However, no differences were observed between control and placebo trials . Men  were moderately faster than women  following the ingestion of coffee between men and women  in salivary caffeine levels between baseline and post-drink following the ingestion of coffee. In contrast, only moderate and small increases were observed during CON  and PLA . Furthermore, no significant difference in salivary caffeine levels was observed between men and women  and PLA . Furthermore, no significant differences between CON and PLA were observed . Blood lactate values were higher in men compared with women  and PLA . However, the magnitude was similar to the difference between CON and PLA . Furthermore, there were no significant differences in blood glucose concentrations between trials  and 1.2%  compared with PLA and CON, respectively. The ergogenic benefits of caffeine ingestion are well documented, e.g.,  recentlyWhilst there were no significant differences in FAS between the trials, FAS did significantly increase over time throughout all trials. These increases in FAS have been demonstrated in previous studies that have employed increasing levels of high-effort exercise . FurtherCYP1A2 isoform of cytochrome P450, with some evidence of downstream enzymatic differences between sexes [Although the primary purpose of this study was not to determine the mechanism of coffee action, it is worth speculating on the underlying mechanism. The metabolism of caffeine is largely dependent on the en sexes , althougen sexes . Adenosien sexes . Howeveren sexes . When spen sexes . Furtheren sexes , which men sexes ,19,22, sThe present study is not without limitations. Whilst the use of a 24 h dietary record to ensure pretrial standardisation is an acceptable method and a good reflection of standard nutritional practice, there may be some concern about its accuracy. A possible limitation is that caffeine consumption was restricted for only 12 hours. However, Irwin et al.  conclude-1 of caffeine increased salivary caffeine levels and improved 5 km cycling time trial performance in men and women by a similar magnitude, which suggests that recreationally active men and women respond similarly, and positively, to coffee ingestion prior to exercise. Furthermore, these results indicate that coffee ingestion may be a practical source of caffeine prior to a 5 km cycling time trial for men and women.Ingesting coffee providing 3 mg·kg


Escherichia coli mutants that had decreased frequencies of colony formation but could grow in liquid medium from a temperature-sensitive mutant collection. Mutations were identified in fabB, which is essential for the synthesis of long unsaturated fatty acids. We then constructed a fabB deletion mutant in a wild-type background. Detailed behavioural analysis of the mutant revealed that under fatty acid-limited conditions, colony formation on solid media was more sensitively and seriously impaired than growth in liquid media. Furthermore, growth under partial inhibition of fatty acid synthesis with cerulenin or triclosan brought about similar phenotypes, not only in E. coli but also in Bacillus subtilis and Corynebacterium glutamicum. These results indicate that fatty acids have a critical importance in colony formation and that depletion of fatty acids in the environment partly accounts for the low frequency of bacterial colony formation.Most bacterial cells in nature exhibit extremely low colony-forming activity, despite showing various signs of viability, impeding the isolation and utilization of many bacterial resources. However, the general causes responsible for this state of low colony formation are largely unknown. Because liquid cultivation typically yields more bacterial cell cultures than traditional solid cultivation, we hypothesized that colony formation requires one or more specific gene functions that are dispensable or less important for growth in liquid media. To verify our hypothesis and reveal the genetic background limiting colony formation among bacteria in nature, we isolated  Bacteria, are the dominant cellular life forms on Earth, with enormous diversity in genes and bioactive metabolites. For over a century, microbiologists have used a pure culture technique based on colony formation to isolate novel micro-organisms. However, numerous studies have reported that only a tiny fraction of bacterial cells living in nature can form colonies on agar plates [Micro-organisms, particularly those in the domain r plates . This loTo isolate novel bacteria, various improvements in culturing conditions have been developed, e.g. diluting nutrients for culturing oligotrophs , removinet al. invented a reliable method for microscopically counting viable cells in a marine sample [In 1979, Kogure e sample . Marine Escherichia coli mutants that could not form colonies but grew in liquid media.Because DVC indicates the number of cells capable of cell division in liquid culture, liquid cultivation may yield many more bacterial cell cultures than traditional plating methods. Indeed, numerous studies have reported that liquid cultivation typically yields higher cultivation efficiencies than solid cultivation, which has sometimes enabled the isolation of novel bacteria from various natural habitats without colony formation . These rE. coli mutant collection [E. coli mutants that cannot form colonies but grow in liquid culture at high temperatures, which might explain the gap in culturability between liquid and solid media. We successfully obtained colonization-defective E. coli mutants that grew in liquid media. Detailed characterization of these mutants revealed that colony formation requires more fatty acids on solid media compared to the requirements in liquid media. Moreover, we experimentally demonstrated that these results can be extended to wild-type E. coli and possibly other bacteria, indicating an important role for fatty acids in bacterial colony formation.In this study, we attempted to screen colonization-defective mutants that retained culturability in liquid media from a temperature-sensitive llection . TemperaE. coli K-12 wild-type strain used in this study was MG1655. An E. coli temperature-sensitive (ts) mutant collection derived from PA3092 [+ strain, PA200, with a plasmid library of E. coli open reading frames (ORFs) [Bacillus subtilis 168 and Corynebacterium glutamicum ATCC13032 were provided by Dr Hirofumi Yoshikawa (Tokyo University of Agriculture) and Dr Saori Kosono (The University of Tokyo), respectively. The Escherichia coli was cultivated in l-broth containing 1.0 % Bacto tryptone , 0.5 % Bacto yeast extract and 0.5 % NaCl, or M9 minimal medium containing 0.6 % Na2HPO4, 0.3 % KH2PO4, 0.1 % NH4Cl, 0.1 % NaCl, 1 mM MgSO4 and 0.1 mM CaCl2, with the addition of 0.5 % casamino acids and 0.4 % d-glucose or 0.4 % d-galactose, which are referred to as M9CAGlc and M9CAGal, respectively. For PA3092, 40 µg ml−1 thymine was added to the l-broth. The Bacillus subtilis was cultivated in SPMM medium containing 1.4 % K2HPO4, 0.6 % KH2PO4, 0.2 % (NH4)2SO4, 0.1 % sodium citrate dihydrate, 0.02 % MgSO4 and 50 µg ml−1 tryptophan, with the addition of 0.5 % casamino acids and 0.5 % d-glucose, which is referred to as SPMMCAGlc. The Corynebacterium glutamicum was cultivated in CM2B medium containing 1.0 % HiPolypepton , 1.0 % Bacto yeast extract and 0.5 % NaCl. To prepare the 1.5 % solid medium, agar powder  was used for the screening of ts mutants, Bacto agar (BD Biosciences) was used for c.f.u. and MPN (most probable number) measurements, and Agarose L03  was used for the other experiments.The m PA3092  and an Fs (ORFs) , were gi−1 before addition to the culture medium. The final concentrations of these solutions in the medium were 0.01, 0.075, 1.0 and 20 µg ml−1, respectively. Palmitoleic acid  or cis-vaccenic acid  was dissolved in 1 % Triton X-100 at 2000 µg ml−1, and then diluted 100-fold with medium, giving a final concentration of 20 µg ml−1. l-Arabinose  was dissolved in sterile water at 200 mg ml−1 and diluted with medium, giving final concentrations of 50, 125, 250, 500, 1000, 2000 and 4000 µg ml−1, respectively. Cerulenin  was dissolved in 50 % ethanol at 2, 4, 6 or 8 mg ml−1 and then diluted with medium, giving final concentrations of 20, 40, 60 and 80 µg ml−1, respectively. For E. coli, triclosan  was dissolved in 50 % ethanol at 5, 10, 15 or 20 µg ml−1 and then diluted with medium, giving final concentrations of 50, 100, 150 and 200 ng ml−1, respectively. For B. subtilis, triclosan  was dissolved in 50 % ethanol at 25, 50, 75, 100, 125 or 150 µg ml−1 and then diluted with medium, giving final concentrations of 250, 500, 750, 1000, 1250 and 1500 ng ml−1, respectively. For C. glutamicum, triclosan  was dissolved in 50 % ethanol at 100, 200, 300 or 400 µg ml−1 and then diluted with medium, giving final concentrations of 1, 2, 3 and 4 µg ml−1, respectively. Ampicillin sodium  was dissolved in sterile water at 2, 4, and 6 mg ml−1 and then diluted with medium, giving final concentrations of 20, 40 and 60 µg ml−1, respectively.Oleic acid  was dissolved in 1 % Triton X-100  at 1, 7.5, 100 or 2000 µg mll-broth in microtitre plates. Each cell suspension was diluted 7500-fold with 150 µl l-broth, and aliquots of approximately 2 µl were inoculated using a Vaccu-Pette/96  into both l-agar plates and 150 µl of l-broth in microtitre plates. After incubation without shaking for 2 days at 41 °C, only one clone grew in the l-broth, while it did not grow on l-agar at 41 °C. To confirm the phenotype of this candidate, after overnight culture at 30 °C, the cells were serially diluted 30-fold with 150 µl of l-broth, and a 2-µl aliquot of each diluted solution was then inoculated onto l-agar and into l-broth. After incubation for 2 days at 41 °C, the clone exhibited a threefold discrepancy between colony formation on l-agar and turbidity formation in l-broth.Colonization-defective ts mutants were obtained as follows. The actual temperature of the sample plates was determined with a temperature logger . A total of 1432 clones from the ts collection were grown overnight at 30 °C in 150 µl E. coli ORF set [l-agar at 41 °C was complemented, i.e. colonies were only formed by a plasmid carrying fabB. PCR amplification and sequencing of this fabBts gene (fabB-1) revealed a C653-to-T transition in the 1221 bp ORF, corresponding to a change of Ala218 to Val in the FabB protein.The ts mutation was mapped by complementation after the conjugative transfer of a plasmid library carrying an  ORF set . The temfabB mutant isolation, we screened an additional 1167 clones from the same ts collection, although this selection was performed at a more moderate temperature of 39 °C. After overnight pre-culture at 30 °C, each clone was serially diluted 30-fold in 150 µl l-broth, and 2-µl aliquots were then inoculated into both l-agar and l-broth. After incubation for 2 days at 39 °C, the colony formation on solid medium and the turbidity in liquid medium were compared. Three clones were isolated as having defects in colony formation. Mapping and sequencing of the three genes responsible for ts revealed that they included the second tsfabB gene (fabB-2) with a single mutation of G95 to A, corresponding to a change of Gly32 to Glu in FabB.To confirm the reproducibility of the fabB ORF of E. coli MG1655 was replaced with the ORF of the Tn5-derived kanamycin phosphotransferase gene by homologous recombination using a pRed/ET Recombination system , which yielded the fabB-deletion strain ∆fabB. Based on previous reports of fabB mutants, ∆fabB was constructed in the presence of 200 µg ml−1 oleic acid in the medium [fabB gene accompanied by its 41 bp 5′ untranslated region was inserted into the NheI site of the mini-F plasmid pKLJ12 [fabB, in which fabB was under the control of the araBAD promoter.The e medium . The fabd pKLJ12 , yieldinCulturability on solid medium and in liquid medium were compared by determining c.f.u. and MPN, respectively. When determining the c.f.u., we did not use a glass spreader for the plating to avoid sample loss and the accompanying experimental error. Instead, cells were inoculated as follows. Bacterial cell suspensions were diluted with saline, and a 100 µl aliquot of each suspension and 650 µl of sterile water were added to a plate and spread on the entire surface by tilting the plate. The sterile water was added so that cells would spread to a sufficient extent over the entire surface of the plate. The plates were sealed with Parafilm and incubated at 30, 37 or 39 °C. c.f.u. values were expressed as colony-forming cell numbers per millilitre of the original culture. MPN was determined as follows. Bacterial cell suspensions were diluted appropriately with liquid medium, and 150 µl of each suspension was then dispensed into a 96-well microtitre plate. The plate, covered with a lid, was sealed with Parafilm and incubated at 30, 37 or 39 °C without shaking. The number of turbid wells per 96 wells was determined; the MPN was then calculated according to the Poisson distribution  and exprfabB were cultured overnight in M9CAGlc medium containing 20 µg ml−1 oleic acid. The cells were washed with saline, and 100 µl of each diluted solution was then inoculated onto several solid medium plates and into liquid medium, followed by incubation at 37 °C; the experimental media contained corresponding concentrations of oleic acid. At each sampling time, all cells were harvested from each plate with sterile water, and an appropriate volume was then spread on an l-agar plate containing 20 µg ml−1 oleic acid to count the proliferating cells per plate, as illustrated in Fig. S1 (available in the online version of this article). Similarly, an aliquot of the liquid medium was harvested and spread to count the number of proliferating cells per millilitre of liquid medium.To determine the number of cells capable of proliferating in solid and liquid media, strains MG1655 and ∆Escherichia coli MG1655 cells grown to OD660=0.8 were washed twice with saline, resuspended in sterile water to give a cell density of approximately 106 cells ml−1, and then incubated at 4 °C. The total cell count was determined with a Multisizer 3 Coulter Counter . The viable cell count was determined with a LIVE/DEAD BacLight Bacterial Viability kit  according to the manufacturer’s recommendations. c.f.u. was determined with l-agarose or M9CAGlc-agarose plates with or without 20 µg ml−1 oleic acid after cultivation for 3 days at 25 °C.2PO4 and 1.5 mM KH2PO4), vortexed and sonicated for 1 min. Soil particles were removed by paper filtration, and the supernatant was spread onto plates. For cultivation, 1/10 strength l-broth solidified with 1.5 % Agarose L03  was used. A mixture of palmitoleic acid  and oleic acid , dissolved in 1 % Triton X-100  at 2000 µg ml−1 each, was added and diluted 100-fold in medium as appropriate. Glycerol  was added to the medium at a final concentration of 100 µg ml−1 as appropriate. After incubation for 10 days at 25 °C, the c.f.u. g−1 soil was calculated from the number of visible colonies formed on the solid medium.Soil was sampled from the forest floor at a depth of approximately 20–30 cm at the University of Tokyo Tanashi Forest, Nishi Tokyo, Tokyo, Japan. The soil was divided into 1 g portions and stored at −80 °C. One gram of soil was suspended in 10 ml phosphate-buffered saline  of interest were essential or that the mutants, if any, were leaky, with only decreased rather than eliminated colony-forming frequencies. We then attempted to obtain mutants with decreased colony-forming frequencies from a collection of ts E. coli strains [We first attempted to isolate colonization-defective mutants that only grew in liquid culture from a transposon-mutagenized library of  strains . These tfabB, which encodes 3-oxoacyl-(acyl carrier protein) synthase I, a key enzyme involved in the synthesis of long unsaturated fatty acids [fabBts mutants, clone 1 and clone 2, are referred to as fabB-1 and fabB-2, respectively (see the Methods section for a detailed description of the mutations). We are currently characterizing the genes responsible for ts and their effects for clones 3 and 4, which proved to have mutations on different genes related to folic acid metabolism.Among the 1432 ts strains in the collection, only 1 clone capable of growing in liquid medium at 41 °C was identified, which is referred to as clone 1. Screening of the other 1167 ts strains at a more moderate temperature of 39 °C revealed 3 clones capable of growing in liquid medium, which are referred to as clones 2–4. After mapping  and sequty acids . Hereaftl-agar (l-broth-based agar) and liquid l-broth. At 39 °C the c.f.u. value of these mutants was approximately 1000-fold (fabB-1) or 100-fold (fabB-2) lower than the MPN value; in contrast, no difference was observed at 30 °C or at either temperature for the parental strain , both abundant unsaturated fatty acids in E. coli, were as effective as oleic acid (18 : 1 ω9c) in supporting the growth of strain ∆fabB . We did not determine the effect of stearic or palmitic acids because of the low solubility of these saturated fatty acids, even with the use of 100 µg ml−1 Triton X-100 as a solubilizer.Palmitoleic acid (16 : 1 ω7c) and fabB cells proliferated slowly, forming invisible microcolonies on the plates. To evaluate the actual proliferation of strain ∆fabB in solid or liquid culture, we harvested cells from solid and liquid media after various incubation times and counted the cells by plating them with 20 µg ml−1 oleic acid (fabB cells could not proliferate well on l-broth-based agarose (l-agarose), but proliferated in liquid l-broth, reaching more than 105 cells ml−1 without fatty acid supplementation  and added specific amounts of oleic acid , the c.f.u. of wild-type E. coli was more than 100-fold lower than the MPN  in E. coli  and has [E. coli . In thisE. coli, B. subtilis and C. glutamicum were also treated with triclosan in solid and liquid cultures at sub-lethal concentrations. Again, we observed serious impairment in colony formation compared to growth in liquid culture .fabB mutants can not only be extended to wild-type E. coli, but also to other bacteria, including B. subtilis and C. glutamicum: the depletion of fatty acids significantly reduces culturability on solid medium compared to that in liquid medium.Based on these results, our findings for E. coli. After prolonged incubation under stressed conditions, such as starvation, E. coli cells enter a distinct static state. Such cells subjected to prolonged stresses form few colonies, even on rich medium, despite retaining various signs of viability [−1 oleic acid supplementation . The addition of unsaturated fatty acids reproducibly increased the c.f.u. by approximately eightfold , which encodes 3-hydroxydecanoyl ACP dehydrase in the fatty acid synthesis pathway [We successfully obtained  culture 3. Gene f pathway .E. coli mutant to the wild-type strain, and furthermore from E. coli to distantly related bacteria, B. subtilis and C. glutamicum. We showed that sub-lethal concentrations of cerulenin inhibit colony formation to a greater extent than is seen in liquid cultivation, and not only for E. coli, but also for B. subtilis .The chemical inhibition of fatty acid synthesis with cerulenin or triclosan expanded the results obtained from an subtilis . E. coliynthesis . Cerulenynthesis . Bacilluis, FabI , the col culture . We obseutamicum . In addiutamicum . Thus, iE. coli cells required fatty acids for higher colony formation , suggesting that the addition of fatty acids does not impair the diversity of bacteria. We avoided the use of Tween 80 or related ester-type detergents because they could supply fatty acids when bacteria secreted esterases.The results obtained from cells defective in fatty acid synthesis by mutation or the addition of drugs were expanded to wild-type cells under starvation. We observed that starved ormation , suggestormation . This refabB mutants and showed interesting differences in growth between solid and liquid cultures in wild-type strains under fatty acid depletion conditions. The molecular mechanisms underlying the prominent effect of unsaturated fatty acids remain unclear. It is likely that bacterial cells under starvation conditions require additional fatty acid-associated processes to initiate colony formation on solid culture compared to in liquid culture. Many bacteria in the natural environment may conserve energy by lowering the level of fatty acid synthesis at the expense of colony-forming ability.In this study, we revealed the critical importance of fatty acids in colony formation in an explicit way by isolating and characterizing E. coli to shift down to a low colony-forming state in response to starvation [Based on the same philosophy as in the present work, in which the low colony-forming state is supposed to be a bacterial genetic response to starvation, we previously took an alternative approach to screen candidates for genetic players in regulating colony formation under the starvation condition . In thisarvation . AlthougClick here for additional data file.


Although parasitic isopods can negatively affect the reproduction and ingestion of several commercially important crustaceans, little is known regarding the mechanisms that underlie these effects.Palaemonetes sinensis infected with the parasitic isopod Tachaea chinensis.In the present study, the iTRAQ (isobaric tags for relative and absolute quantification) approach was applied to identify differentially expressed proteins in the Chinese grass shrimp T. chinensis infection. The differentially expressed proteins revealed that parasitism may inhibit the immune response, thereby increasing host vulnerability to additional lethal infection. Furthermore, T. chinensis may secrete anticoagulants to inhibit hemolymph clotting. Moreover, the isopod parasite placed a heavy metabolic burden on the host, particularly with respect to glucose metabolism.On the basis of our analysis, we identified 1262 proteins from a total of 4292 peptides. There was a significant difference in the expression of 182 proteins between the control and infected groups, among which 69 were upregulated and 113 were downregulated after Our study is the first to use the iTRAQ-based proteomic approach to analyze the effects of an isopod parasite on its host. The results we obtained using this approach will make a valuable contribution to understanding the molecular mechanisms underlying isopod parasitism on crustaceans. Parasitic isopods have a negative impact on a variety of commercially important fish and crustacean hosts . The rapEctoparasitic isopods feed primarily on blood (hemolymph) that oozes from wounds of the host after the integument has been perforated by the isopod’s mandibles, which can cause host stress, tissue damage, secondary infection and mortality , 8–10. ATachaea chinensis, one of the common ectoparasites of economic shrimps, is widely distributed in China and neighboring countries [Tachaea chinensis is approximately 0.8 cm long, mainly attaches to the ventral thoracic region of shrimps and can readily be detected by the naked eye after parasitization  used in this study were collected from a rice field in Panjin City, Liaoning Province, China, in April 2018, and transported to the aquaculture laboratory at Shenyang Agricultural University. Palaemonetes sinensis (3.48 ± 0.35 g) were caught using a net cage in a lake nearby the laboratory (no isopod parasitism had previously been reported in this area). These shrimps were acclimated in two 300-l square fiberglass recirculation tanks. Each tank was linked to a circular flow system. The water temperature was maintained at 22 ± 0.5 °C and the photoperiod was set at a light:dark cycle of 12:12 h. After 2 weeks of acclimation, 20 healthy P. sinensis individuals  were transferred into individual plastic tanks (15.8 cm diameter and 32.1 cm height), each of which contained 5 l of water from the acclimation tanks, and environmental conditions were the same as those used for the acclimation period. Subsequently, 10 T. chinensis specimens were transferred into 10 tanks separately (one per host) and formed the infected group. The shrimps in the remaining 10 tanks were used as the controls. According to the results of our previous study, most parasitized shrimp began dying after 15 days (unpublished data); therefore, after 7 days in the present study, all 10 control shrimps and all 10 infected shrimps (without parasites) were placed separately in twenty 2-ml RNAse-free tubes that were immediately frozen in liquid nitrogen for storage until protein extraction.The g for 15 min and washed with 200 µl of UA buffer. After incubation in 100 µl of 50 mM iodoacetamide in UA buffer for 30 min in the dark, the samples were centrifuged again at 14,000× g for 10 min. The filters were washed three times with 100 µl of UA buffer and then twice with 100 µl of dissolution buffer. Finally, 2 μg of trypsin  was added to each filter and digested overnight at 37 °C.For each sample, 200 µg of protein was diluted in 200 µl of uric acid (UA) buffer  and transferred onto a 10-kDa ultrafiltration filter. The samples were centrifuged at 14,000× iTRAQ labeling was performed using an iTRAQ Reagent-8 plex Multiplex Kit according to the manufacturer’s instructions . The proteins in the control group were labeled with reagent 118, 119 and 120, whereas those in the infected group were labeled with reagent 121, 122 and 123.The iTRAQ-labeled samples were purified by strong cation exchange (SCX) chromatography using a Polysulfoethyl 4.6 × 100 mm column  in an AKTA Purifier 100 . After SCX fractionation, 33 fractions were collected and combined into 10 pools and desalted on C18 cartridges . Each fraction was segregated using a nano HPLC Easy nLC system  incorporating two Thermo Fisher Scientific  EASY columns . The flow rate was set at 250 nl/min. The peptides were eluted with a gradient of mobile phase A (0.1% formic acid in water) for 5 min, and then in a gradient starting from 0 to 35% mobile phase B (0.1% formic acid in 84% acetonitrile) for 45 min, followed by an 8 min linear gradient to 100%. Finally, the samples were maintained in 100% mobile phase B for 2 min. Each sample was subjected to mass spectrometry (MS) survey using a Q-Exactive mass spectrometer (Thermo Finnigan). Briefly, a full MS survey scan was performed for a mass range of 300 to 1800 m/z with resolution of 70,000 at m/z 200. High energy collisional dissociation (HCD) fragmentation was used for MS/MS, and the 10 most intense signals in the survey scan were fragmented. Normalized collision energy was set at 30 eV with a 0.1% underfill ratio.The raw files were analyzed by using Proteome Discoverer v.1.4 software . Identification of the proteins was conducted using the MASCOT v.2.2 search engine . The protein identification and quantitation parameters are shown in Table These proteins were linked to the following databases for downstream analysis: Quick GO , KOG (Clusters of orthologous groups for eukaryotic complete genomes) and KEGG (Kyoto Encyclopedia of Genes and Genomes).T. chinensis (0.88 ± 0.15 cm) and P. sinensis (3.61 ± 0.29 g) were collected in November 2018. After acclimation for 2 weeks, each of the 50 shrimps in the experimental group were artificially infected with a single isopod and randomly divided into five groups. One shrimp was collected from each group at 0 (control), 24, 48, 72, 96 and 120 h after the artificial infection; from these, 200 μl of hemolymph was extracted and divided into two aliquots. Each 100-μl subsample was centrifuged for 10 min  and the resulting supernatants were immediately used for the determination of hemocyanin content and CCO activity. We detected no significant difference among groups with regard to shrimp weight.The proteome data were confirmed at both the transcriptional and protein levels. At the transcriptional level, the significantly expressed proteins were compared with the expression of their coding genes in our previous transcriptome data. Moreover, 55 shrimps were used to validate the proteome by using the activity of cytochrome c oxidase (CCO) and hemocyanin content: 5 for the control and 50 for the experimental group. The 2, 50 mmol/l Tris-HCl, pH = 8.0) and readings were taken at 334 nm (O.D.334). The concentration of hemocyanin was calculated using the following equation: hemocyanin content (mg/ml) = 2.33 × O.D.334 × 100. CCO activity was measured using a CCO testing kit  according to the manufacturer’s guidelines.The hemocyanin measurements were performed spectrophotometrically. The supernatant samples were diluted with 1.9 ml of buffer .Statistical analyses were performed using analysis of variance (ANOVA). Significant differences between means were determined by Duncan’s test at a significance level of The iTRAQ analysis of the present study showed 17,131 queries in the 162,387 spectra. The protein database contained a total of 41,095 protein sequences, and the fragment and peptide mass tolerances were set to ± 20 ppm and ± 0.1 Da, respectively. Among these, a total of 1262 unique proteins were identified across the 4292 peptides  were identified between the control and infected groups. Among these differentially expressed proteins, 69 were upregulated and 113 were downregulated after T. chinensis infection  shared the same annotation with transcripts. Among these 20 proteins, 16 (80%) showed expression trends similar to the transcriptome expression trends in the control and infected group, including nine upregulated and seven downregulated proteins/genes  = 21.21, P = 0.002); 1.57 ± 0.42  = 40.97, P < 0.001); 1.21 ± 0.27  = 20.54, P = 0.002); 1.87 ± 0.48  = 40.71, P < 0.001); and 2.01 ± 0.55  = 72.41, P < 0.001) mg/ml at 24, 48, 72, 96 and 120 h after isopod parasitism, respectively. Compared with the control group (0 h), the activity of CCO significantly increased by 7.57 ± 0.88%  = 84.98, P < 0.001); 4.31 ± 0.46%  = 16.99, P = 0.003); 16.45 ± 2.54%  = 189.89, P < 0.001); and 20.17 ± 3.26%  = 169.89, P < 0.001) in shrimps parasitized for 48, 72, 96 and 120 h, respectively.The results of our analysis of hemocyanin content and CCO activity over a 120 h period after infection are shown in Fig. T. chinensis has resulted in heavy economic losses of commercially farmed crustacea, particularly in Penaeus vannamei, Macrobrachium nipponense, Exopalaemon carinicauda and Palaemonetes sinensis [Investigation of the response to different pathogens represents an important research goal for elucidating immune and related components in the defense system of crustaceans –20. In rsinensis –23. TherT. chinensis could use its mandible to feed, suck the hemolymph and promote hematological changes in P. sinensis. Therefore, similar to other blood-feeding parasites, T. chinensis may also be implicated in the transmission of blood-dwelling diseases between shrimps, which we intend to examine in our future research.Hemocyanin has been studied in several host-pathogen interactions, which have indicated that hemocyanin also plays an important role in the immune system of shrimps , 24–26 aT. chinensis may secrete various anticoagulants, which could work cooperatively and prevent blood clotting by downregulating the expression of clotting-related proteins at the site of injury.Most invertebrates need to rapidly prevent the loss of blood or equivalent fluids through inflicted injuries . In shriT. chinensis parasitization [T. chinensis may have to balance between evading the host immune response, similar to acanthocephalans parasitizing gammarids [Moreover, we found that five actin proteins and a Toll-like receptor protein were significantly downregulated. In contrast, these proteins have been found to be upregulated during viral and bacterial infection . Crustactization . Parasitammarids , 38, andNephrops norvegicus [According to the KEGG analysis performed in the present study, two pathways involved in glucose metabolism, namely carbon metabolism and glycolysis/gluconeogenesis, were among the top five statistically significant different pathways. Moreover, proteins associated with these two glycometabolism-related pathways, including crustacean hyperglycemic hormone (CHH), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and carbohydrate sulfotransferase (CST), were significantly upregulated. These results are similar to those obtained from our corresponding transcriptome analysis . Carbohyrvegicus . HoweverT. chinensis could inhibit the hemolymph clotting system of P. sinensis during parasitization. Moreover, isopod parasites place a heavy metabolic burden on their hosts, particularly with regards to glucose metabolism. Overall, our study provides valuable empirical data that will support future molecular research on isopod parasitization of crustaceans.To the best of our knowledge, this is the first study to investigate the response of shrimps to an isopod parasite using an iTRAQ-based proteome method. The differentially expressed proteins related to hemocyanin content and the hemolymph coagulation system were identified as downregulated after isopod infection. This may indicate that, similar to blood sucking parasites, Additional file 1: Figure S1. Picture of T. chinensis attached to the ventral thoracic region of P. sinensis.Additional file 2: Table S1. Total proteins compared to GO, KOG, and KEGG databases.Additional file 3: Table S2. Lists of differentially expressed proteins.Additional file 4: Figure S2. GO enrichment analysis.Additional file 5: Figure S3. KEGG enrichment analysis.


A general protocol is developed to obtain D‐glucosamine from three widely available biomass residues: shrimp shells, cicada sloughs, and cockroaches. The protocol includes three steps: (1) demineralization, (2) deproteinization, and (3) chitin hydrolysis. This simple, general protocol opens the door to obtain an invaluable nitrogen‐containing compound from three biomass residues, and it can potentially be applied to other chitin sources. White needle‐like crystals of pure D‐glucosamine are obtained in all cases upon purification by crystallization. Characterization data  of D‐glucosamine obtained from the three chitin sources are similar and confirm its high purity. NMR investigation demonstrates that D‐glucosamine is obtained mainly as the α‐anomer, which undergoes mutarotation in aqueous solution achieving equilibrium after 440 min, in which the anomeric glucosamine distribution is 60% α‐anomer and 40% β‐anomer. Nitrogen is also required to make amino acids that are the building blocks of proteins, the essential catalysts, and structural components of all plant and animal cells. bond −94 kJ mol−13) and ammonium ion (NH4+) at mild temperatures. NH3 and NH4+ along with nitrites (NO2−) and nitrates (NO3−), which are produced from nitrification reactions  are water soluble and reactive species that can be more easily assimilated by biological systems.At high temperatures, the bond between the two nitrogen atoms breaks to produce reactive nitrogen species that combine with other elements, such as oxygen and hydrogen, to form N‐containing compounds that can be absorbed by living organisms. In the atmosphere, lightning and meteor trails are hot enough to produce measurable amounts of biologically useful N‐containing compounds that are eventually brought down to living organisms by rain.Since the invention of agriculture, humans have realized that harvesting continuously removes “fixed” nitrogen from the soil. Therefore, nitrogen has had to be supplied somehow to the soil to keep up with food demand. The first nitrogen source used by humans to enrich the soil was manure, followed by guano (pigeon's droppings) and saltpeter (chiefly composed of sodium and potassium nitrate) extracted from natural reservoirs in the Pacific Islands and in the deserts of Chile, respectively. However, it was not until the development of the first industrial process to convert nitrogen gas from the air into ammonia by the German scientists Fritz Haber and Carl Bosch in the 1900s that nitrogen fertilizers became readily available in large scale for food production.N‐acetyl glucosamine subunits linked together through covalent β(1 → 4) glycosidic bonds  were collected from local markets in the state of Sao Paulo, Brazil. The definition of shrimp shells is understood here as shrimps' abdominal segments, carapace, antennae, and tail. Chemical composition of shrimp shells includes 15%–25% chitin, 30%–40% minerals, 30%–50% proteins and pigments, and 0.3%–1% lipids.Shrimp shells from Whiteleg shrimp (Quesada gigas) were collected from trees located at the University of Campinas campus  during October 2015 and October 2016 (Spring), and used without any previous treatment.Cicadas can be found in regions with temperate to tropical climates. Cicadas molt their cuticles leaving their sloughs behind attached on trees. Cicada sloughs consist of ≈37% chitin, 40% proteins, 12% minerals, 3% lipids, and 8% water.Periplaneta americana) were handpicked in random locations in Campinas, São Paulo, Brazil, and used with no previous treatment.Cockroaches can be found throughout the world as the species can withstand a wide range of environments, from hot subtropical deserts to the extremely cold temperatures of the Artic. Due to their global availability and abundance, cockroaches are an interesting source of chitin as their exoskeleton and wings contain about 13%–18% of the biopolymer. Nymph and adult American cockroaches .Proteins, pigments, and lipids are removed by treating the previously demineralized chitin source with 1.0 2.3Chitin hydrolysis is carried out with concentrated HCl (37%) at 90 °C, for 2 h. Two distinct hydrolysis events occur during this step : i) cleavage of the glycosidic bonds, depolymerizing chitin into its monomeric units, and ii) cleavage of the amide bonds that lead to deacetylation of the glucosamine subunits. Crystallization using ethanol/water affords white needle‐like crystals of pure D‐glucosamine hydrochloride, which are filtered off and dried under vacuum.TableChitin content on the three biomass sources as well as the average yields of the obtained glucosamine is gathered in 2.41H, 13C, COSY), IR, and mass spectrometry. As expected, characterization data for glucosamine obtained from the three chitin sources are similar.Glucosamine obtained from the three biomass residues was characterized by NMR  with the expected isotopic distribution  that are attributed to the proton linked to the anomeric carbon  J‐coupling values vary between 8 and 14 Hz.J = 3.6 Hz while the peak at 4.94 ppm has J = 8.4 Hz. Therefore, the two doublets at 5.45 and 4.94 ppm correspond to the protons linked to the anomeric carbon in α and β‐anomers, respectively. The other nuclei on the glucosamine hydrochloride structure resonate at the expected chemical shifts and multiplicity . Data show that at the first time point (20 min) glucosamine existed as 80% α‐anomer and 20% β‐anomer. As time elapsed, the amount of β‐anomer increased at the expense of the α‐anomer. The mutarotation process reached equilibrium at t = 440 min, in which the anomeric glucosamine distribution was about 60% α‐anomer and 40% β‐anomer . Mutarotation occurs via ring opening after protonation of the ring oxygen affording the aldehyde acyclic intermediate. The carbonyl group in the open structure can suffer conformational change through rotation of the C—CO bond, resulting in conversion of anomers after ring closing.3A general protocol was optimized to obtain the invaluable nitrogen‐containing compound D‐glucosamine from three widely available biomass residues: shrimp shells, cicada sloughs, and cockroaches. The protocol includes three steps: (1) demineralization, (2) deproteinization, and (3) chitin hydrolysis. Upon purification by crystallization, white needle‐like crystals of pure glucosamine were obtained in all cases. NMR, IR, and MS data of glucosamine obtained from the three chitin sources are similar and demonstrate its high purity. This process does not obey all the principles of Green Chemistry because it uses corrosive HCl and NaOH. However, when analyzing the green metrics of this organic synthesis protocol several advantages can be highlighted4NMR: 1H and COSY NMR spectra were recorded at 400.0 MHz and 25 °C on a Bruker AC 400 spectrometer. 13C NMR spectra were acquired at 100.0 MHz and 25 °C on a Bruker AC 400 spectrometer. All chemical shifts are reported in ppm relative to the solvent residual peak.Mass Spectrometry: The mass spectrum was obtained using a Waters Micromass Quattro Micro API instrument using electrospray ionization and triple quadrupole mass analyzer. The sample was analyzed by direct infusion with methanol/water/formic acid 49.9:50:0.1 (v/v). Experimental conditions: Capillary voltage: 3 kV; cone voltage: 15 V; extractor voltage: 3 V; radio frequency lens: 0.5 V; source temperature: 150 °C; desolvation temperature: 200 °C; desolvation gas flow: 800 L h−1; and cone gas flow: 50 L h−1.Infrared Spectroscopy: Infrared spectra were recorded using an Agilent Cary 630 Fourier‐transform infrared spectroscopy (FTIR) spectrometer with the attenuated total reflectance accessory.General Procedure for Demineralization: The chitin source was stirred in 700 mL of the aqueous HCl 0.5 m solution at room temperature for 3 h. The solution was filtered and the solid was washed thoroughly with distilled water, and dried .General Procedure for Deproteinization: The demineralized solid was introduced in a 1 L round‐bottom flask. 400 mL of a NaOH 1.0 m aqueous solution was added, and the reaction mixture was submitted to reflux during 2 h. The solution was filtered, and the obtained chitin was washed with distilled water, and dried .General Procedure for Chitin Hydrolysis to Obtain D‐Glucosamine Hydrochloride: Chitin was introduced in a 500 mL round‐bottom flask, and 80 mL of concentrated HCl (37%) was added. Under stirring, the solution was heated at 90 °C for 2 h. In this step, chitin dissolved and the solution color became brownish. The solution was cooled to 60 °C and 10 mL of distilled water was added. Activated charcoal was added, and the solution was stirred at 60 °C for 30 min. The solution was filtered twice through paper. Ethanol was added and the solution was kept in the fridge to allow crystallization of glucosamine hydrochloride. The white crystals were filtered, washed with cold ethanol, and dried under high vacuum .1H NMR : 5.46 , 4.96 , 3.94–3.68 , 3.55–3.47 , 3.31 , 3.02 . 13C NMR : 92.7, 89.2, 76.2, 72.0, 71.6, 69.7, 69.6, 60.5, 60.3, 56.7, 54. ESI‐MS (M)+m/z calcd for C6H14NO5+: 180.09, found 180.21. FTIR (cm−1): 3346, 3278, 3091, 3031, 2841, 1611, 1583, 1533, 1417, 1387, 1248, 1181, 1092, 1027, 997, 914, 882, 851, 771, 577, 544, 498.The authors declare no conflict of interest.SupplementaryClick here for additional data file.


The purpose of this study has been to determine the effect of acute consumption of noni and chokeberry juices vs. energy drinks on blood pressure, heart rate, and blood glucose. The subjects divided into 4 groups, which consumed three portions of noni or chokeberry juices  and energy drink (ED) or water (200 mL) at one-hour intervals. All participants had their blood pressure (BP), both systolic and diastolic BP (SBP and DBP), as well as heart rate (HR) and blood glucose (BG), measured. Consumption of noni juice caused a significant decrease in SBP and DBP of 5.0% and 7.5%, respectively, while, the consumption of chokeberry juice slightly decreased only DBP by 3.6%. On the contrary, consumption of three portions of EDs caused a significant increase in DBP by 14.7%. The BG of participants consuming noni juice decreased by 7.3%, while the consumption of EDs increased BG by as much as 15.8%. Acute consumption of noni juice contributed to a significantly decreased SBP, DBP, and HR as well as a mild reduction of BG. Consumption of chokeberry juice caused only a slight reduction of DBP. Contrary to juices, EDs consumption resulted in an increase of blood pressure  and blood glucose. The results of the study showed that noni juice may be effective in lowering blood pressure and blood sugar levels, but there is a need to continue research on the long-term effect of this juice. Morinda citrifolia L.) originates from Southeast Asia (Polynesia and India), but it is also grown in South America and the Caribbean Islands [Morinda citrifolia as one of the dietary components which have a positive impact on the cardiovascular system, especially preventing hypertension, atherosclerosis, and dyslipidemia [The fruit of noni , most probably owing to the high content of polyphenols . ConversThe purpose of this study has been to determine the effect of acute consumption of noni and chokeberry juices vs. energy drinks on arterial blood pressure, heart rate, and blood glucose.The study was carried out on a group of volunteers, students of a private college . Healthy volunteers (aged over 18 years) were recruited for the study. Exclusion criteria were as follows: cardiovascular disease, any medication affecting the cardiovascular system, diabetes, other chronic diseases, pregnancy, lactation, and regular alcohol intake. All subjects signed an informed consent after reading the purpose and schedule of the research. Originally, 96 volunteers were recruited. The participants were asked to abstain from drinking alcohol or taking any supplements (for at least 24 h prior to the experiment) and to exclude from their diet (for at least 12 h before the test) sources of caffeine such as coffee, tea, and cola type of drinks. In addition, the research participants were requested not to do any excessive physical activity prior to the test. Before the tests, all subjects were informed about the research objective and schedule, after which they signed a written consent form to participate in the study. Finally, 88 subjects took part in the study. Eight people did not participate . Subjects were divided into 4 groups: 1st—consuming 30 mL noni juice (noni group); 2nd—consuming 200 mL chokeberry juice (chokeberry group); 3rd—consuming 200 mL energy drink (ED group); 4th—drinking 200 mL water (control group). The assignment of participants to each group was random, aided by a random number generation formula (an MS Excel application). Before the onset of the tests, the participants received verbal instructions.Organic noni fruit and chokeberry juices, both a rich source of polyphenols, were used for the study. The juices were cold-pressed from whole fruits. Chokeberry originated from Poland, while noni came from French Polynesia. Juices underwent mild flow pasteurization (the temperature not exceeded 80°C) in order to preserve nutrients. The ED group was given to consume one of the most popular energy drink, which contained 32 mg of caffeine per 100 mL. The energy drink was purchased in a local grocery shop, but prior to the test, it was coloured with a food dye and got rid of carbon dioxide. The control group was served still water which had also been coloured with a food dye. Until the tests, all the drinks  were stored at room temperature. Moreover, juices and energy drinks were previously analyzed to determine their pH and antioxidant capacity (TP and DPPH). The pH of the tested samples of juices was measured with a glass electrode at room temperature. Antioxidant capacity and total polyphenol content assays were described in previously studies . The infThe study was carried out between May and June 2018. On the day of the tests, all participants had fasted measured blood glucose, blood pressure (systolic and diastolic), and heart rate. Next, each subject consumed three portions of the drinks (described in the Samples section) at one-hour intervals. The noni group drank 30 mL of NJ ; the chokeberry group consumed 200 mL of ChJ ; the ED group had 200 mL of energy drink ; and the control group consumed 200 mL of water . Each participant had their blood pressure (SBP and DBP) and heart rate (HR) measured four times, i.e., before having the first juice or ED and 1, 2, and 3 h after the consumption of each portion. BP and HR measurements were done in accordance with the guidelines of the European Society of Hypertension and European Society of Cardiology , and thep values lower than 0.05 were considered as significant.All the results were statistically analyzed by calculating the mean and standard deviation. The interpretation of the results was performed in MS Excel 2010 Analysis ToolPak software , with one-way analysis of variance (ANOVA) and Tukey's test as the post hoc test; The tests were performed on young adults , most having a normal BMI (79.5% of the participants). 15 subjects (17%) were overweight, and 3 people were underweight. Baseline characteristics of the study participants are shown in Prior to the tests, all participants were interviewed about their dietary habits, including daily intake of caffeinated drinks, juices, and nectars. The results showed that most of the subjects consumed fruit juices (53 people consumed fruit juices daily). Forty-nine subjects drank coffee every day, of which 23 people preferred brewed coffee and 19 chose instant coffee. Twenty-eight subjects admitted to regular drinking energy drinks and cola-type beverages.p=0.024) by 5.0%, i.e., down to 113.6 mm·Hg (p=0.006), from 77.0 to 72.0 mm·Hg, i.e., by 6.5%, after drinking three portions of NJ (p=0.030) relative to the initial value . Slightly greater decrease owing to the consumption of chokeberry juice was observed in DBP, which was lowered by 1.8, 3.3, and 3.6%, respectively, after the first, second, and third portion of the juice. However, the decrease from 84.0 to 81.0 mm·Hg after drinking a total of 600 mL of chokeberry juice was not statistically significant (p=0.304) (p=0.507) decrease, as they fell down from 79.3 to 76.6 beats per minute.The chokeberry group (similar to the noni group) was observed to have decreased SBP and DBP as well as HR. The SBP decreased by 2.1%  after consumption of the first portion of chokeberry juice . The subp=0.304) . The valp=0.206) from 119.2 to 124.8 mm·Hg (4.8%), while the DBP increased significantly (p < 0.001) from 73.9 to 84.8 mm·Hg (14.7%) (p=0.243) increase, rising from 76.9 to 80.8 beats per minute, even after consuming three portions of EDs.Unlike the noni group and chokeberry group, the group consuming ED experienced an increase in the SBP, DBP, and HR after each portion of energy drink. Initially, having drunk the first portion of energy drink 200 mL), the participants had an unchanged SBP, while their DBP increased by 4.2% Figures  and 2. A00 mL, th (14.7%) . The heap=0.014) increase in BG, by 15.8%, i.e., from 83.6 to 96.8 mg/dL. In the control group (consuming water), the level of BG did not undergo any significant change (p=0.226).Further analyzes focused on the blood glucose levels. Consumption of three portions of NJ (each at 30 mL) caused decrease in BG by 7.3%, from 86.1 to 79.8 mg/dL . Having p=0.024 and p=0.006) decrease in systolic and diastolic blood pressure, by 6.0 and 5.0 mm·Hg, respectively. Consumption of three portions  of chokeberry juice resulted in a small decrease in SBP and DBP by 1.3 and 4.0 mm·Hg, respectively, which was not statistically significant (p=0.766 and p=0.304). In another pilot, crossover study [n = 6) and older adults (n = 7) received in random order, either a single 300 mL dose or 3 × 100 ml doses of high-flavonoid cherry juice provided at 0, 1, and 2 h. Two hours after consumption of 1 × 300 mL of the juice, a decrease of SBP and DBP by 5.5 mm·Hg was observed, while the HR decreased by 4.8 beats per minute. The group consuming 3 × 100 mL of the juice was reported to have a decrease in SBP by 3.5 mm·Hg, DBP by 3.3 mm·Hg, and HR by 3.3 beats per minute. Blood pressure returned to the initial values 6 hours after drinking the juice. More favourable effects were observed in the first group, who drank a larger amount of juice on a single dose [p > 0.05), while DBP decreased from 89.0 ± 10.9 to 82.9 ± 6.2 mm·Hg (p < 0.05) [In our study, acute consumption of NJ  caused a significant  . In our  < 0.05)  conducte < 0.05) . In our  < 0.05) , intake  < 0.05)  noted th < 0.05) . Moreove < 0.05) –33.In our research, consumption of three portions of noni or chokeberry juice decreased the HR by 5.8 and 2.7 beats per minute, respectively. In another study participated by young adults, 2 hours after drinking 300 mL  of juice rich in flavonoids, the heart rate decreased by 4.8 or by 3.3 beats per minute .Contrary to the juices, increased SBP and DBP and HR were observed after consumption of three portions of EDs, by 5.6 and 10.9 mm·Hg, respectively, and by 3.9 beats per minute. In another study, higher SBP and DBP were also noticed 2 hours after drinking 355 mL of ED (114 mg of caffeine). The values increased by 5.2 and 6.1 mm·Hg, respectively . There wStudies on animals have also shown the efficiency of using NJ in lowering BG. Nayak et al.  have shop=0.754) lower level of BG, whereas Skoczyńska et al. [p < 0.05) BG decrease, from 99.3 ± 14.5 to 91.8 ± 14.3 mg/dl. Other extracts containing flavonoids, especially anthocyanins, can decrease the BG. A study conducted on 10 overweight (BMI ≥ 25) people, aged 18–65 years, who consumed a drink made from acai berry in an amount of 100 g twice a day for 1 month, were found to have a BG lowered from 98.0 mg/dl to 92.8 mg/dl [Lycium barbarum L.) to rabbits with diabetes  and hyperlipidemia resulted in a significant decrease in BG after ten days of experiment. The treatment also lowered total cholesterol and triglycerides in blood plasma but raised the level of HDL-C [In our study, the subjects drinking chokeberry juice were noticed to have an only slightly , caused a considerable decrease in SBP, DBP, and HR, while inducing only a slight decrease in BG in young adults. Doses of 30 ml of noni juice are often recommended and have been used in previous studies , 9, 41. We have demonstrated that acute consumption of noni juice contributes to a significant decrease in both systolic and diastolic blood pressures as well as heart rate in young healthy adults. Moreover, NJ caused a slight decrease in the blood glucose. Consumption of chokeberry juice resulted in a decrease of DBP, but the changes were not statistically significant. The changes in SBP and HR were also insignificant. No statistically significant changes in BP and HR were observed in the control group consuming water. Contrary to the juices, the energy drinks contributed to a significant increase in DBP and BG but did not have a significant influence on SBP. Thus, consumption of EDs may induce an adverse impact on human health, whereas juices rich in polyphenols may play a beneficial role, especially by decreasing arterial blood pressure and blood glucose. These findings require further studies to explain the influence of long-term consumption of noni and chokeberry juices on the above parameters.


The 3-PCBs and 6-DDTs levels were slightly higher in leaf vegetables compared to root vegetables. The detection of PCBs and DDTs in the vegetables suggest the probable use of PCBs containing pesticides. Although the observed concentrations were below the WHO maximum residue limits, consumption of such contaminated leaf and root vegetables portend a health risk.Persistent organic pollutants (POPs) are dangerous and toxic pollutants that may cause adverse effects on human and animal health, including death. POPs such as polychlorinated biphenyls (PCBs) and pesticides are subtly released into the environment from industrial and agricultural use. Global circulation is due to their trans-boundary transport capacity, contingent on aerodynamic and hydrological properties. Plants have capacity to take-up POPs, and these bio-magnify along heterotrophic transfer pathways. In this study, levels of selected 6-PCB congeners and 3- DDTs in some leaf and root vegetables were investigated. Leaf and root vegetables were collected from different horticultural farms areas in Cape Town. The 6-PCBs and 3-DDTs were recovered from the samples using solid phase extraction(SPE), followed by GC-MS analysis. The ΣPCBs and ΣDDT (on-whole basis), were ranged: 90.9–234  They may also be unintentionally, generated as by-products of activities involving combustion1.Persistent organic pollutants (POPs) such as pesticides and polychlorinated biphenyls (PCBs), are synthetic substances produced for a wide range of sanitary health, agricultural and industrial uses3. Ritter et al.4 reported that, compounds such as pesticides and PCBs can persist and bio-accumulate by factors up to 70,000 fold in the environment. Low vapour pressure POPs can volatilize into air at ambient temperatures, and undergo transboundary atmospheric transfer reaching far destinations, or re-condensed at cooler temperatures.Nearly all POPs are characterized by poor water solubility, hence their resistance to decomposition. This results in their environmental persistence over a long time. They may therefore accumulate in soil, partitioned between water and sediment, or exponentially bio-accumulate up in the food chain7. For instance, some pesticides have been reported to induce endocrine disruption with consequences on reproductive system, sex-linked disorders, and shortened lactation in nursing mothers’9. Carcinogenic and mutagenic responses11, as well as certain neurophysiological effects, such as attention deficits, learning disorders, behavioral problems including increased aggressivity, and poor gross and fine motor coordination have been linked to foetal exposure to POPs12.Exposure to low concentrations of POP compounds can lead to severe toxic consequences and adverse health effects, such as cardiovascular effects (damage to immune and respiratory systems), damage to critical organs, teratogenic effects (birth defects), miscarriages and even death in humans and wildlife14. While many of these chemicals pose serious environmental and human health risks on their own, the cumulative health effect that a combination of these chemicals may have is potentially of greater concern116.Although different types of POPs exist, the polychlorinated biphenyls (PCB) and the organochlorine pesticide (OCP) compounds are among the most persistent, capable of magnification and with high mammalian toxicity. Polychlorinated biphenyls (PCBs) consist of 209 low vapour pressure, heat resistant, non-flammable congeners with high dielectric constants, used as heat exchange fluids or as insulating medium in electrical transformers and capacitors. Of these number, 13 congener compounds exhibit dioxin-like properties, and are reported to show extremely high toxicityet al.17 soil-to-plant transfer is one of the major pathways of pesticides transport to shoots i.e. leaves and stems of plants grown on contaminated soil, or on soils irrigated with contaminated wastewater. There is therefore need to be concerned about the exposure and levels of PCBs and pesticides in vegetables, since they are a major diet and a transfer pathway.Persistent and bio-accumulative pesticides include many first generation organochlorine (OC) compounds such as 1, 1′-bis(4-chlorobenzene) (DDTs), dieldrin, toxaphene and chlordane. These compounds were largely used in agriculture for pests and weeds control, in order to maximize produce yield and thereby increase economic gains on the long run. Consequently, farm produce such as vegetable, perennial crops and many other pesticide-sprayed plants, take–up pesticides, storing them in plants cell lipid membrane, passing them from one heterotrophic level to another. According to Khan et al.18 revealed that dietary intake of vegetables grown in the vicinities of thermal power plants is associated to carcinogenic health risks. Since vegetables can be potentially contaminated, dietary exposure could therefore elicit toxic responses, causing adverse effects including human and animal ill health and even death.Aside from the compromise of habitats and environment health, is the concern over the potential impact PCBs and DDTs can infringe on the world economy through food contamination. As these substances move up the food chain, their concentrations increase (bio-magnify), therefore animals atop food chain  may be exposed to several folds magnified concentrations of these chemicals. Findings from a study” on “Incremental Cancer Risk Assessment” conducted by Khillare 19. A strategy for human and animal protection is continuous environmental and food monitoring. While many analyses on water and fatty foods such as fish, milk, cheese and some groceries have been reported previously24, information concerning pesticide residues in short shelve life fresh vegetables and other food crops is scanty. Bouwman25 reported that despite the risks, they pose to human health, which are of great public concern information about the levels, distribution and fate of many POPs in soils, vegetables and other food samples is limited. Also, the assessment of POP residues especially the PCBs and OCPs in vegetables and other biological matrices in many Africa countries are limited, hence the scarcity of data on their levels, characterization and safety/health impactIn 2012, the United Nation Environment Programme (UNEP) Scientific Committee of Experts (SC), targeted 22 POPs including the dirty dozen , mirex, toxaphene, PCDD/Fs i.e. polychlorinated dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs) and polychlorinated biphenyls (PCBs)) for elimination or restricted use, in order to minimize the contamination of food chainIn this study the levels of 6-PCBs congeners:  and 3-DDTs , 4, 4′-dichlorodiphenyldichloroethylene (DDE) and 4, 4′-dichlorodiphenyldichloroethane (DDD)) residues were investigated in fresh leaf  and root vegetables  using GC-MS. This is in order to evaluate and characterize plant food  health status vis-a-viz their occurrence levels, as well as set a benchmark in policy for periodic monitoring and guideline thresholds for human, animals and environmental health and safety.Acetonitrile, acetone and acetic acid of analytical grade, purity > 98% from Sigma Aldrich used for the analysis. High purity standards (≫99.9%) used for instrumental calibration were purchased from Restek Inc. Internal standards, deuterated pp’DDT-d8 was purchased from Sigma Aldrich. Pre-packed solid phase cleanup kits (50 mL Teflon centrifuge/extraction tube and 15 mL Teflon centrifuge/clean-up frits tubes containing polymeric reverse phase (PRP) column) were obtained from phenomenex.µg/mL by diluting 1 mL, 1000 µg/mL in 100 mL volumetric flask. Thereafter, cocktail of working calibration standards of 25, 50, 100, 500 and 1000 ng/mL were prepared by successive  dilution in 100 mL volumetric flask to obtain a linear regression of five-point multi-component calibration standards of 8-mix. At initial, the standards of each of the analytes of the 6-PCB congeners and 3-DDTs were individually injected in six replicate measurements each to determine their retention time and characteristic fragment patterns. Thereafter, cocktail of each concentration levels of the working standards was injected on-line the GC-MS in selected reaction monitoring (SRM) mode. The integrated peak areas  in response to gradient of standards of each of the measured analytes (chromatogram) was used in the identification of the analytes, while quantitation was by external calibration.Stock solutions of PCBs_110, _118, _138, _149, _153, _180, DDT, DDE and DDD standards were prepared to achieve 10 et al.26. According to Sloan et al.27 an internal standard recovery of 60–120% should be achieved for analytes intended for GC-MS analyses.Also, about 200 µL of surrogate internal standard solution of 4, 4′-DDT_d8 at low, medium and high concentrations was added to about 200 mL, 5% methanol in deionized water. The surrogate spiked solutions were allowed to equilibrate and treated using the same sample treatment procedures. Each concentration level was prepared and injected in triplicate as recommended by Camino-Sanchez Analytical homogeneity (<15% RSD) of the sample results was tested by investigating the reproducibility of replicate measurements of samples at specified intervals/frequency, (10 samples), and that of replicate measurements of certified reference materials (CRM-IAEA-140-OC) during analysis.Spinacia oleracea, cabbage – Brassica oleracea, lettuce – Lactuca sativa, dhanial – Coriandrum sativum, celery – Apium graveolens, parsley – Petroselinum crispum, and kale – Brassica oleracea), and root vegetables , were collected from seven formal and informal farms geo-referenced 18.5304588 E and –34.0154588 SS, around Cape Town, between January and October 2017 on a monthly basis.Farm soils, leaf vegetables  fired (550 °C for 1 hr) dilute nitric acid pre-cleansed/swabbed aluminum foil; root vegetables were collected by slicing into furnace fired dilute nitric acid pre-cleansed aluminum foils; and the soils were pooled from farm top soil (0–15 mm) into nitric acid pre-cleansed aluminum foil using a stainless steel hand trowel. Each of the samples collected, were then placed in well-labelled zip plastic bags in order to isolate the sample from each other to prevent cross contamination, and thereafter place in a cooler with Ice Park for onwards transfer to the laboratory. The collected samples were frozen at −20 °C in the refrigerator until processing. All samples were process within 48 hr. of collectionThe leaf and root vegetables were split into two; with a split washed in clean water before air-drying, while the other split was dried directly without washing and dried at ambient laboratory conditions. The farm soil samples were also air dried in the laboratory at ambient conditions. After drying, each of the leaf and root vegetables were crushed into fines, while the soil samples were pulverized to <2 mm particle size.μL of internal standard (10 ppm) was added to the homogenate, followed by 10 ml of Milli-Q-water and 10 ml of acetonitrile acidified with 10% of acetic acid. The mixture was thoroughly homogenized on a vortex at a revolution of 2000 rpm for 2–3 min. The mixture was allowed to stand for 15 minutes. Thereafter, approximately 6 g MgSO4 and 1.5 g NaCl was then added to the homogenate, and thoroughly mixed and vortexed for another 3 minutes. The homogenate mixture was allowed to stand for 5 min, and then centrifuged at a revolution of 2 000 rpm for about 5 minutes. The PCBs and DDT mass extract in acetonitrile supernant was decanted from the solid residue and cleaned up in the PRP pre-packed cleanup column. The recovered extract in acetonitrile was concentrated <0.5 mL using the CentriVap concentrator under nitrogen stream and reconstituted to 1 mL in acetonitrile for analysis.Extraction of the analytes was carried out using the pre-packed solid phase extraction column. About 5 g of homogenized vegetable sample was weighed into centrifuge tube. Thereafter, 100 4 and 1 g NaCl were added to the homogenate afterwards, and vortexed again for another 2 min. About 20 mL of mixture of high purity acetonitrile and acetone (60:40) was then added to homogenate, and left to equilibrate on a horizontal shaker for 10 min, and afterwards allowed to stand for 20 min. The PCBs and DDT mass extracts in acetonitrile and acetone mix was decanted from the soil residue, and concentrated to near dryness using a CentriVap concentrator under nitrogen stream, and reconstituted to 1 mL in acetonitrile for analysisAbout 5 g each of the pulverized soil sample was weighed into centrifuge tube. This was followed by the addition of mixture of 10 mL of Milli-Q-water and 10 mL of acetonitrile acidified with 10% of acetic acid. The resulting mix was homogenized on a vortexed at 2000 rpm for 2 min. Approximately 5 g MgSOCongener specific analysis was conducted on the recovered analytes in acetonitrile-acetone extracts, respectively pooled from the fresh leaf and root vegetables, and top soil samples to identify and quantify residues of PCBs: 110 , 118 , 138 , 149 , 153 , and 180 , and DDTs: dichlorodiphenyltrichloroethane , dichlorodiphenyldichloroethylene , and dichlorodiphenyldichloroethane .μm film thickness), at a flow rate of 3 mL/min, with nitrogen as make-up gas. The injector port temperature was kept at 250 °C, while the sample streams over the column in gradient of temperature set initially at 70 °C and maintained for 2 min holding time. The temperature was afterwards ramped at a rate of 25 °C/min to 180 °C, held for 3 min; 15 °C/min to 250 °C, held for 2 min, and then 8 °C/min to 290 °C, held for 5 min.Analysis were performed using a gas chromatograph  fitted with an auto sampler and coupled a mass spectrometry detector (5975) (GC-MSD). About 1.0 μL each of the samples were injected in the splitless mode, in-stream of helium carrier gas over a DB-5 (5%-phenyl-95%-dimethylpolysiloxane) capillary column  quadrupole via the transfer line set at a temperature of 300 °C. Ionization source temperature was set at 300 °C, and operated in the negative electrospray ionization (ESI), with argon collision scanning, operated in the selected reaction monitoring (SRM) mode at capillary voltage set at 3 kV, while the ions data were acquired via selected ion monitoring (SIM) with two characteristic ions. Quantification of the analytes was performed using the external standard technique.The data obtained were subject to evaluation using descriptive statistics and one-way analysis of variance (ANOVA) with consequent Duncan test . The results were interpreted based on homogeneous groups at p < 0.05 significance level28. Validation parameters such as limit of detection (LOD) and limit of quantification (LOQ) of the method were evaluated using blank and fortified replicates (n = 10) for all analytes in each matrix mix.The efficiency of the GC-MS method for the determination of the 6-PCBs and 3-DDTs was validated based on the guidance criteria of the International Conference on HarmonizationThe retention time and their SRM m/z characteristic fragmentation pattern of each of the individual analyte was determined by means of six online injections. The MS/MS chromatograms for the elution of each of the injected individual analytes: 4.4′-DDE, 4.4′-DDD, 4.4′-DDT, PCB_110, PCB_118, PCB_138, PCB_149, PCB_153 and PCB_180 indicated their retention time (supplementary file).The retention time for the elution of each of the 6-PCBs and 3-DDTs analytes, and their characteristic SRM m/z fragmentation pattern for the qualifying and quantifying ions, parent and product masses as well as their collision energies were recordedng/mL. The coefficient of regression (R2) obtained for the concentration intervals between 25 ng/mL and 1000 ng/mL were all greater the 0.00 (R2 > 0.99)  of experimental blank (noise) with respect to the slope (m) of calibration curve of gradient concentration plot ng/mL) of PCB_110, PCB_138, PCB_118, PCB_149, PCB_153, and PCB_180 were: 0.075, 0.018, 0.034, 0.022, 0.022 and 0.027 respectively, while the LOD of 4, 4′-DDD, 4, 4′-DDE, and 4, 4′-DDT were 0.052, 0.028, and 0.016 ng/mL respectively (supplementary file).The LODs for each analyte was thereafter obtained as the quotient of three multiples of the standard deviation . The average recovery of the labelled internal standard 4, 4′-DDT_d8 ranged between 72% and 90%, while those for the 3-DDTs and 6-PCBs ranged between 68 and 94%. Intra-class correlation (ICC) used for the comparison of influences of intra-individual and inter-individual variances in the recoveries of analytes in spiked and unspiked samples, on the whole variance observed for the DDTs and PCBs measurements were in agreement with the of The Two-Way Random Absolute ICC values.The concentration profiles of the 6-PCBs congeners and the 3-DDTs in the leafy vegetables, root vegetables and farm soils varied. This could be attributed to several factors including selective soil abstraction and translocation, variation in aerials uptake based of amount reaching plant aerial parts, stability of PCB and DDTs, pesticide dissipation and degradation  in soil, biological factors such as microbial population, types, and characteristics, weather and soil condition. The concentrations (on-whole basis) of the residues of the 3-DDTs and 6-PCBs in the leafy vegetables was found to be generally variable Table .Table 2Cng/g and 14.9 ± 5.89 ng/g, respectively, while celery vegetable had the highest concentration of 4, 4 – DDT (28.6 ± 9.81 ng/g)  concentrations was observed in parsley, with mean concentration of 22.0 ± 7.80 3DDTs was observed in spinach , while the highest was noted in celery . The observed low concentrations may be a consequence of the ban on the use of DDT OCPs. However, DDT somehow finds its way into the South African market for crops and animals protection purposes, as well for residual indoor spraying to control vector29. Aside from this, the occurrence of DDT in vegetables may also be due to aerial deposition of leaks and other usage source dissipated airborne suspended DDTs, on soil and vegetation, especially in near vicinities where soil is intensively used for farming, and their persistence (owing to their hydrophobic properties) as a result of usage deposition from the past.The lowest concentration of Σng/g; PCB_118, 18.8 ± 10.4–27.9 ± 12.7 ng/g; PCB_138, 12.1 ± 7.70–19.9 ± 8.42 ng/g; PCB_149, 17.0 ± 7.29–23.9 ± 7.28 ng/g; PCB_153, 16.6 ± 5.75–24.6 ng/g and PCB_180, 13.6 ± 4.73–23.4 ± 8.02 ng/g in all the tested leafy vegetables  was found to be the highest while the concentrations of PCB_138 congener  was found to be the lowest.The concentrations of PCB_110 congener in all leafy vegetables  of 4, 4-DDT in the root vegetables i.e. carrots, spring onion, leek, turnip, reddish, broccoli and cauliflower were: 31.4 ± 9.94, 26.2 ± 8.75, 24.2 ± 9.03, 22.6 ± 7.38, 21.8 ± 5.32, 20.1 ± 8.19 and 19.6 ± 6.86, respectively. The levels (ng/g) of metabolites 4.4-DDD were: carrots, 20.1 ± 5.09; spring onion, 18.9 ± 5.21; leek, 16.5 ± 4.71; turnip, 15.6 ± 3.18; broccoli, 14.6 ± 3.07; reddish, 12.7 ± 4.18 and couli flower, 11.2 ± 2.55; and 4.4-DDE were: carrots, 14.7 ± 2.95; spring onion, 13.4 ± 3.09; leek, 12.2 ± 3.26; turnip, 12.1 ± 2.72; reddish, 11.9 ± 2.84; broccoli, 11.6 ± 2.19 and cauliflower, 10.4 ± 4.17.  in all the root vegetables tested, while carrots contained the highest Table . The meang/g for PCB_110; 18.9 ± 7.25 and 29.1 ± 9.07 ng/g for PCB_118; 12.9 ± 8.42 and 18.9 ± 8.01 ng/g for PCB_138; 16.1 ± 8.84 and 22.7 ± 9.74 ng/g for PCB_149; 15.6 ± 5.87 and 22.2 ± 9.95 ng/g for PCB_153 and 13.4 ± 7.35 and 21.2 ± 9.31 ng/g PCB_180, respectively, in cauliflower and carrots (Table 6PCBs) reached 843 ng/g, and was slightly elevated in comparison to the sum of the measured DDT and its DDD and DDE metabolites (Σ3DDT), which total, 362 ng/g concentration. The observed concentrations of the 3-DDTs and 6-PCBs in the different root vegetables though variable, are consistent with findings from other studies32. The observed mean concentrations of the POPs in leaf vegetables were generally slightly higher than in the root vegetables, except for DDE.The mean concentration of the 6-PCB congener were ranged between: 13.6 ± 9.61 and 35.6 ± 12.8 The concentrations of the 3-DDTs and 6-PCBs in farm soils also varied across all 7-farms (Table 2 > 0.53 – ϒ2 > 0.78) between DDTs and PCBs levels detected in each of the root vegetable types and soil concentration levels. Multivariate regression model revealed also strong relationships between ΣDDTs and ΣPCBs concentration levels in the farm soils, farm site locations, and environmental pollution.There were strong correlations . This suggests that the vegetables may probably have retention holding capacities that could facilitate ease of plant contamination vis-a-viz uptakes. Although, the differences in the levels observed in the leaf and root vegetables are not significant (p < 0.05), the detected concentrations were significantly lower than observed in the farm soils. It is however not clear, whether the total ΣDDTs and ΣPCBs are at levels that could initiate any stress on the plants, or even toxic responses. This depends on factors such as the magnitude of contaminants concentration, age of DDTs and PCBs in the soils, and plants uptake and accumulation capacity. The C-C, C-H as well as C-Cl intra-atomic bonds in many DDTs and PCBs confers on them such properties that facilitates their high molecular weight, low vapour pressure, low polarity, low water solubility, resistance to hydrolysis, and poor microbial activities. This may therefore result in their environmental stability/persistence with a potential to induce toxicity on different organisms35 and also governs the dynamics of their uptake pattern.Comparison of leaf and root vegetables concentration of the 3-DDTs and 6-PCBs, revealed a slightly higher levels in all leaf vegetables than in root vegetables; with mean concentrations of PCB 110; PCB 118, PCB 138, PCB 149, PCB 153 and PCB 180; 28.6 37. According to Polder et al.38 the ratio between 4, 4 – DDT and its metabolites 4, 4 – DDE and 4, 4 – DDD “is often used to describe recent or historic exposure of DDT product to the environment”. Low ratios of 4.4 – DDE/4.4 – DDT and 4.4 – DDD/4.4 – DDT in different matrices is indicative of recent use of DDT, high ratios of 4.4 – DDE/4.4 – DDT and 4.4 – DDD/4.4 – DDT indicate the occurrence of aging DDT after long time use, with increase in residues of the adduct metabolite products of environmental decomposition. The ratio of 4.4 – DDE/4.4 – DDT and 4.4 – DDD/4.4 – DDT in the leafy vegetables ranged between 0.59 in cabbage and dhanial, and 0.72 in spinach, and 0.43 in parsley and 0.61 in lettuce, respectively. In the root vegetables, the ratio of 4.4 – DDE/4.4 – DDT and 4.4 – DDD/4.4 – DDT were ranged 0.59 in turnip and spring onion, to 0.72 in broccoli and cauliflower, and 0.43 in leek to 0.61 in radish. The 4.4 – DDE/4.4 – DDT and 4.4 – DDD/4.4 – DDT ratio observed in the leaf and root vegetables suggest slight aging from possible recent use.Since DDTs and PCB congeners show resistance to breakdown, the metabolism of these substances is very slow. Thus, the proportion of their metabolites will vary with the parent compounds in different mediaBAF = Cp/Cs; Cp - concentration in aerial part of plant, and Cs - concentration in soil) for the 3-DDTs and 6-PCBs in leaf vegetables ranged from 0.25 for 4, 4-DDT in dhanial and kale to 0.78 for 4, 4-DDD in celery, and from 0.11 for PCB_180 in spinach to 0.71 for PCB_110 in lettuce. The bioaccumulation factor of the root vegetables on the other hand ranged from 0.25 for 4, 4-DDE in spring onion to 0.58 for 4, 4-DDD in leek and 0.13 for PCB_180 in leek and broccoli to 0.66 for PCB_110 in Leek.In addition, the capacity of the leaf and root vegetables to retain the 3-DDTs and 6-PCBs is indicative of their potential as a pathway for the translocation of these substances especially through the food chain. Results indicated variability in the ability of the vegetables to bio-accumulate the DDTs and the PCB congeners. The leafy vegetables bioaccumulation factors  compared with the more chlorinated PCBs (lower BAF). Similarly the bioaccumulation factor for metabolite 4, 4-DDD was higher in all vegetables compared with 4, 4-DDE and 4, 4,-DDT. This could be due to their solubility, though soil concentration levels were quite higher than observed in the leaf and root vegetables. Thus, heterotrophic transfers hold the likelihood of human and animal exposure, and this portends a potential for health compromise.Generally, DDTs and PCB congeners occurred in all the farm soils, with intermittent detection of high concentration levels along discrete farm portions, while their concentration levels in the leaf and root vegetables were low. Our findings showed that vegetables and crop planted in farmlands within locations near residences and urban centers, especially informal farms in homes, may be exposed to leaked pesticides from home use and arrays other contaminants; many of which are classified as endocrine disruptors and neuro-degenerative substances. Therefore, there is a need for control by enacting and implementing regulatory policies that will curtail the use DDTs and PCBs containing products, as well as the release of DDTs and PCBs.39 reported that the occurrence of elevated levels of 4, 4′-DDE and 4, 4′-DDT may be associated or probably due to exposure to DDT contaminated foods, or food prepared using DDT contaminated wood. The higher concentration levels detected in soils may possibly be due to the large commercial and subsistence farming activities.Evidence clearly indicate heterogeneity in the occurrence levels of the 3-DDTs and 6-PCBs detected in at the different farm sites. The concentration observed in the leafy vegetables and the root vegetables were also variable over farms. This might not be unconnected with the differential partition based on the soil characteristics and topographic inequalities of the farm plots. More also, the sources of the 3-DDTs and 6-PCBs into soil and their distribution pattern and pathways as well as uptake mechanism into the vegetables and perhaps other vegetation is not very clear. ChannaThus, the presence of DDT and PCBs in soil could be a good indicator of deposition pattern due to the likely use of pesticides containing OCPs and some PCB congeners, for agricultural purpose such as pest control, increase crop yields, or contamination from long-range transport from diffuse sources afar. However, soil concentration of the sum of 3-DDTs and 6-PCBs were lower than reported in soil samples analyzed in Glasgow, Torino, Aveiro, Ljubljana and Uppsala with concentrations 22.0 µg/kg, 14.0 µg/kg, 7.90 µg/kg, 6.80 µg/kg, and 5.70 µg/kg respectively. Background values of 0.53 µg/kg was reported in upper agricultural field in Germany. The variabilities in the distribution profile of the 3-DDTs and 6-PCBs across the farm land soils, may be associated with the age of application/deposition, vapor pressure/volatilization potential and climatic influence.40. The levels detected in the investigated vegetables in this study were within the global range, with the sum of the concentrations of the 3-DDTs being below the European Commission41 legally tolerated maximum residue level (MRL) of 500 µg/kg pesticide residues in food, leaf vegetables, herbs and edible flowers. The observed levels were consistent with the concentration of OCPs  vegetables such as graviola, mullaca and balsamina in Bolivia and Peru40. An average OCPs concentration range of 0.18 ± 0.14 ng/g to 0.76 ± 0.43 ng/g was reported in India, while PCBs concentration ranged between <DL and 99.4 ng/g (13.4 ± 0.06 ng/g). Study results also agrees with Aichner et al. and Wang et al.43 for plants in the Kathmandu and Tibetan Plateau, China, respectively. Lower concentrations of PCBs were however reported in Vietnam, Romania, China, Mexico47, while higher levels noted in Turkey48.The global concentrations of OCPs in all varieties of plants were estimated to range from 0.5 to 100 µg/kg dry weightng/mL respectively for the 3-DDTs and 6-PCB congeners. The sum of the concentration of the 3-DDTs in the leaf and root vegetables ranged between 41.2–66.1 ng/g, and 38.9–62.1 ng/g, respectively, while the 6-PCBs ranged between 90.9–149 ng/g, and 95.8–234 ng/g, respectively.Concentrations of the 3-DDTs i.e. DDT and its metabolites DDD and DDE and 6-PCBs congeners in the 7-varieties of leafy vegetable and the 7-varieties of root vegetables collected from different farms were variable. The concentrations of these POPs were quantified with an allowed limit of detection (LOD) and the limit of quantification (LOQ) between 0.010–0.022 µg/L and 0.030–0.042 The observed concentrations of the 3-DDTs and 6-PCBs congeners, 110, 118, 138, 148, 153 and 180 were low. The concentrations of the tested POPs in all the leaf and root vegetables were all below the European Commission maximum residue levels and the maximum residue limits suggested by World Health Organization (WHO). Thus, the vegetables are relatively uncontaminated, and the occurrence of residues of the POPs may not be the result of POP pesticides use. The DDTs and PCBs in the vegetables and soil may be due to contamination arising from deposition of long ranged transported pesticide aerosol, incineration or split leaks from vector control residual indoor application from nearby residences and other sources.Supplementary Tables