Functional human studies often faced ethical challenges due to invasive medical procedures; thus, in this study, we implemented PolyFermS fermentation models to mimic the infant proximal colon, which were inoculated with immobilized fecal microbiota of two 2-month-old infants. We investigated the impact of pH, retention time, and accumulation of dl-lactate on microbiota composition and metabolic activity. We found that a drop in pH from 6.0 to 5.0 led to increased LPB and decreased LUB concomitantly with lactate accumulation. Increasing the RT resulted in complete lactate consumption associated with increased LUB. Our data highlight for the first time the impact of key abiotic factors on the metabolism of lactate, which is an important intermediate product for ecology and infant health.The metabolism of lactate is important for infant gut health and may lead to acute lactate and/or H 2 associated with pain and crying of colicky infants. Because gut microbiota studies are limited due to ethical and safety concerns, in vitro fermentation models were developed as powerful tools to assess effects of environmental conditions on the gut microbiota. In this study, we established a continuous colonic fermentation model (PolyFermS), inoculated with immobilized fecal microbiota and mimicking the proximal colon of 2-month-old infants. We investigated the effects of pH and retention time (RT) on lactate metabolism and of lactate-utilizing bacteria (LUB) exhibiting little or no H2 production. We observed that a drop in pH from 6.0 to 5.0 increased the number of lactate-producing bacteria (LPB) and decreased LUB concomitantly with lactate accumulation. Increasing RT from 5 to 10 h at pH 5.0 resulted in complete lactate consumption associated with increased LUB. Supplementation with dl-lactate (60 mM) to mimic lactate accumulation promoted propionate and butyrate production with no effect on acetate production. We further demonstrated that lactate-utilizing Propionibacterium avidum was able to colonize the reactors 4 days after spiking, suggesting its ability to compete with other lactate-utilizing bacteria producing H2. In conclusion, we showed that PolyFermS is a suitable model for mimicking young infant colonic microbiota. We report for the first time pH and RT as strong drivers for composition and metabolic activity of infant gut microbiota, especially for the metabolism of lactate, which is a key intermediate product for ecology and infant health.The metabolism of lactate impacts infant gut health and may lead to acute accumulation of lactate and/or HIMPORTANCE The metabolism of lactate is important for infant gut health and may lead to acute lactate and/or H2 accumulation, pain, and crying as observed in colicky infants. Functional human studies often faced ethical challenges due to invasive medical procedures; thus, in this study, we implemented PolyFermS fermentation models to mimic the infant proximal colon, which were inoculated with immobilized fecal microbiota of two 2-month-old infants. We investigated the impact of pH, retention time, and accumulation of dl-lactate on microbiota composition and metabolic activity. We found that a drop in pH from 6.0 to 5.0 led to increased LPB and decreased LUB concomitantly with lactate accumulation. Increasing the RT resulted in complete lactate consumption associated with increased LUB. Our data highlight for the first time the impact of key abiotic factors on the metabolism of lactate, which is an important intermediate product for ecology and infant health. Bifidobacterium, Lactobacillus, Bacteroides, Streptococcus, Staphylococcus, and Enterococcus , selected for little or no H2 production, for their potential to colonize and metabolize residual lactate. A recent classification of the genus Propionibacterium allocated the cutaneous P. avidum to the new genus Cutibacterium  and LUB strain addition on composition and activity of infant gut microbiota were investigated during period 2. Each period consisted of stabilization with CR control conditions for 11 to 23 days, followed by treatment application for 8 to 14 days.Two PolyFermS continuous fermentation models were used in this study to mimic the conditions in the proximal colon of a 2-month-old formula-fed infant. The fermentation setup consisted of a first inoculum reactor (IR) inoculated with 30% (vol/vol) gellan-xanthan gel beads that immobilized the fecal microbiota, which was connected to a control reactor (CR) and four treatment reactors (TRs). All TRs and the CR were operated in parallel, continuously inoculated with 5% fermentation effluent from the IR, and additionally fed with 95% fresh medium, as presented in Materials and Methods and illustrated in Firmicutes and Bacteroides. Compared to donor 2, the fecal sample from donor 1 harbored higher levels of Enterobacteriaceae , Bifidobacterium (9.2 and 7.6 log gene copies g−1), Veillonella (9.4 and 8.6 log gene copies g−1), and Lactobacillus (9.1 and 6.0 log gene copies g−1).Feces from two 2-month-old healthy infant donors with similar gestational ages, birth weights, ages, and feeding practices see . The two10.1128/mSystems.00264-18.10TABLE S1Table S1, DOCX file, 0.02 MB.(a) Baseline characteristics of donors 1 and 2; (b) primers used for the detection of predominant bacterial groups in fecal and effluent samples by qPCR. Download Copyright © 2019 Pham et al.2019Pham et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Firmicutes, Bacteroides, and Bifidobacterium  and 0.8 log lower in the CR (8.6 log gene copies ml−1) compared to the donor 1 fecal sample (9.4 log gene copies g−1). Also, 1.5- and 2.3-log-higher levels of Veillonella were detected in the IR (10.1 log gene copies ml−1) and CR (10.9 log gene copies ml−1) compared to the donor 2 fecal sample (8.6 log gene copies g−1), respectively.The IR and CR were operated under constant conditions at pH 6.0 with an RT of 5 h during the entire experiment and used for testing stability over 79 and 57 days in F1 and F2, respectively. After the first stabilization time that allowed gut microbiota to colonize the reactors and to reach steady conditions, the microbiota composition of the IR and CR detected by qPCR during both fermentations was very similar to the corresponding donor fecal sample for most of the targeted groups, including acterium . DiffereFirmicutes, Enterobacteriaceae, Bacteroides, Bifidobacterium, Veillonella, and Lactobacillus , lactate, and formate measured with HPLC indicated overall stable microbial metabolic profiles in the IR and CR of both fermentations after an initial colonization and stabilization period of 17 days . During bacillus . The modevotella .10.1128/mSystems.00264-18.1FIG S1FIG S1, TIF file, 2.0 MB.Lactate, formate, acetate, propionate, butyrate, and total metabolite concentration and total carbon number of the inoculum reactor (IR) and control reactor (CR) during fermentations 1 (a and b) and 2 (c and d). B, batch fermentation . Download Copyright © 2019 Pham et al.2019Pham et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mSystems.00264-18.2FIG S210 copy numbers ml−1 fermentation effluent) of specific bacterial groups determined by qPCR in the inoculum reactor (IR) and control reactor (CR) during fermentations 1 (a and b) and 2 (c and d). Data were expressed as average from technical duplicates. Red, Firmicutes; black, Enterobacteriaceae; green, Bacteroides; purple, Bifidobacterium; blue, Veillonella; yellow, Lactobacillus. Download FIG S2, TIF file, 0.6 MB.16S rRNA gene copy numbers  of 16S rRNA genes at genus level analyzed in fermentation effluent using Illumina MiSeq V3-V4 amplicon sequencing in the inoculum reactor (IR) and control reactor (CR) during fermentations 1 (A) and 2 (B). Values <1% are summarized in the group “Other.” When assignment at genus level was not possible, the highest-level taxonomy assignment is shown. Download Copyright © 2019 Pham et al.2019Pham et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the No significant differences in composition and metabolic activity of the microbiota between the CR and TRs after the stabilization period were found in both fermentations .10.1128/mSystems.00264-18.4FIG S4n = 3). Download FIG S4, TIF file, 1.0 MB.Metabolic activity and composition of microbiota in the control reactor (CR) and treatment reactors (TR) at the end of stabilization periods. Values are mean results ± SD , and decreases in propionate (P < 0.001), isobutyrate (P < 0.001), and butyrate (P < 0.001) production at pH 5.0 compared to pH 6.0 were shown (P < 0.01) and significantly decreased propionate, butyrate, and isobutyrate (P < 0.01 for F1 and P < 0.05 for F2) production were measured at pH 5.0 compared to pH 6.0 (P < 0.001) or formate in F2 (P < 0.05) relative to pH 6.0. Significantly lower levels of Veillonella (P < 0.01) and Bacteroides (P < 0.001) and higher levels of Lactobacillus (P < 0.001) and Enterobacteriaceae (P < 0.001) were measured using qPCR for effluent samples at pH 5.0 compared to pH 6.0 when combining data from the two fermentations  and Prevotella  and higher relative abundances of Lactobacillus , Enterococcus , and Bifidobacterium  were recorded for both fermentations at pH 5.0 compared to pH 6.0 using MiSeq; however, no sequencing replicates prevent statistical analysis on MiSeq data (Ruminococcus (0.19% versus 19.3%) and Peptostreptococcaceae (0.19% versus 7.2%) and high relative abundances of Citrobacter (7.6% versus 2.8%) and Enterobacteriaceae (11.0% versus 6.6%) were measured at pH 5.0 compared to pH 6.0 (Collinsella (5.9% versus 29.9%) and Bacteroides (2.3% versus 21.4%) was observed in F2 at pH 5.0 compared to 6.0 . *, P < 0.05; **, P < 0.01; ***, P < 0.001. Download FIG S5, TIF file, 1.3 MB.Effect of pH on the metabolic activity of infant gut microbiota in two PolyFermS models. (a and b) Concentrations of lactate, formate, and SCFA produced at pH 6.0 (black bar) and pH 5.0 (yellow bar) in fermentations 1 (a) and 2 (b). (c and d) Concentrations of lactate, formate, and SCFA produced at pH 6.0 (black bar) and pH 7.0 (yellow bar) in fermentations 1 (c) and 2 (d). Values are mean results ± SD  and an increase in butyrate (P < 0.05) (Enterobacteriaceae (P < 0.001), Firmicutes (P < 0.001), Veillonella (P < 0.001), Bacteroides (P < 0.05), and total bacteria (P < 0.01) at pH 7.0 compared to pH 6.0 (P < 0.01) and Firmicutes and Bifidobacterium levels (P < 0.05) and increased butyrate (P < 0.01) and formate (P < 0.05) accumulation compared to pH 6.0. Lower relative abundances of Bifidobacterium and Prevotella and higher relative abundances of Enterococcus were also observed in both fermentations at pH 7.0 compared to 6.0   and a sio pH 6.0 . The impo pH 6.0 c and d. d to 6.0  and b. FP < 0.001) compared to 5 h with pooled data from both fermentations (P < 0.05) and total metabolite (P < 0.01) levels compared to an RT of 5 h (P < 0.01), Firmicutes (P < 0.05), Enterobacteriaceae (P < 0.01), and Bacteroides (P < 0.001) when pooling data from the two fermentation (Bifidobacterium (P < 0.05) and Veillonella (P < 0.05) in F1 compared to those with a 5-h RT  on the gut microbiota composition and metabolic activity was tested at pH 6.0 in TR2 of both models during experimental period 1 . An RT ontations  and by 4ntations a and b. T of 5 h . A 10-h entation . We alsoa 5-h RT . In contSeq data  and b. Fe 5-h RT  and b.10.1128/mSystems.00264-18.6FIG S6n = 3). *, P < 0.05; **, P < 0.01; ***, P < 0.001. Download FIG S6, TIF file, 1.3 MB.Effect of retention time (RT) on the metabolic activity of infant gut microbiota in two PolyFermS models. Concentrations of lactate, formate, and SCFA produced at 5-h (black bar) and 10-h (yellow bar) RTs at pH 6.0 (a and b) and at pH 5.0 (c and d). Values are mean results ± SD  in both fermentations  and TR4  during period 1, because similar conditions were used for all TRs during the stabilization period, resulting in similar microbiota composition and activities . At pH 5ntations c and d. t pH 5.0 . In contt pH 5.0 .dl-lactate in nutritive medium to mimic the accumulation of lactate in the infant gut resulted in significant lactate accumulation (P < 0.001) as well as an increase in acetate (P < 0.001), propionate (P < 0.001), and total SCFA (P < 0.001) production compared to no supplementation, when combining data from the two fermentations  and F2 (71.5 and 28.5%). No significant effect of lactate supplementation on microbial composition by qPCR was observed  with addition of lactate. In contrast, adding 60 mM dl-lactate appeared to affect microbial relative abundances of some groups, with observed increased Peptostreptococcaceae (10.9% versus 7.1%) and decreased Citrobacter (0.6% versus 3.9%) and Enterobacteriaceae (4.2% versus 11.3%) abundances in F1, and increased Collinsella (43.4% versus 29.3%) and Veillonella (22.8% versus 14.0%) and decreased Bacteroides (5.7% versus 14.1%) and Enterococcus (1.5% versus 8.8%) abundances in F2  in F1 (a) and F2 (b); without (black bar) and with addition of 60 mM dl-lactate and 108 CFU ml−1E. limosum (yellow bar) in F1 (c) and F2 (b); and without (black bar) and with addition of 60 mM dl-lactate and 108 CFU ml−1P. avidum (yellow bar) in F1 (e) and F2 (f). Values are mean results ± SD (n = 3). *, P < 0.05; **, P < 0.01; ***, P < 0.001. Download FIG S7, TIF file, 0.5 MB.Effect of the addition of lactate and lactate-utilizing bacteria on the metabolic activity of infant gut microbiota in two PolyFermS models. Concentrations of lactate, formate, and SCFAs produced without (black bar) and with addition of 60 mM Copyright © 2019 Pham et al.2019Pham et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the P. avidum or E. limosum was selected among infant LUB for its capacity to utilize lactate with no or little H2 production, respectively. We tested the impact of daily spiking with each strain individually at a high cell concentration (108 CFU ml−1) in reactors supplemented with 60 mM dl-lactate to mimic lactate accumulation.E. limosum was detected at high levels in F1 and F2  in reactors 22 h after spiking (E. limosum and lactate supplementation led to significant increases in lactate (P < 0.01), formate (P < 0.05), propionate (P < 0.001), and total SCFAs (P < 0.001)  in F1 and propionate  in F2 compared to respective stabilization. This treatment also decreased Enterobacteriaceae detected by qPCR in F1 (P < 0.05)  . The impntations  and d. E < 0.05) , which wuring F2 .Propionibacterium, we quantified P. avidum by specific plating of effluent samples of all reactors during stabilization, after daily spiking with 1 × 108 (TR3) or 5 × 108 (TR4) CFU ml−1P. avidum and during the washout period after the treatment during F2 . P. avidum reached similar high cell counts of 8.6 ± 0.9 log CFU ml−1 (TR3) and 8.4 ± 0.1 log CFU ml−1 (TR4) for the two addition levels. Interestingly, 4 days after the last P. avidum addition, significantly higher levels were detected in TR3 and TR4 by plate counts  compared to nontreated reactors  .Due to the lack of specific primers for uring F2 . Before 10.1128/mSystems.00264-18.8FIG S810 CFU ml−1 fermentation effluent) of P. avidum measured by plate counts of reactor effluents before, during, and after supplementation with P. avidum in F2. Yellow and orange bars indicate reactors that were spiked with 108 CFU ml−1 (TR3) and 5 × 108 CFU ml−1 (TR4) P. avidum, respectively, during treatment. Values are mean results ± SD (n = 3). (b) P. avidum in reactor effluents compared to theoretical washout curves before, during, and after supplementation with 108 CFU ml−1 (left) and 5 × 108 CFU ml−1 (right) P. avidum in F2. Download FIG S8, TIF file, 0.3 MB.(a) Viable counts , acetate (P < 0.001), propionate (P < 0.001), and total metabolite (P < 0.001) production when combining data from the two fermentations (P < 0.001), decreased Enterobacteriaceae (0.5 log copy number), and increased E. limosum (1.5 log copy number) (Veillonella (P < 0.01) and decrease in E. hallii (P < 0.05) levels after the addition of P. avidum and lactate compared to stabilization with this treatment  . Combinireatment . Trends uring F1 .dl-lactate, the addition of 108 CFU ml−1P. avidum together with 60 mM dl-lactate led to a decrease in lactate (P < 0.001) and an increase in E. limosum (P < 0.05) (8 CFU ml−1E. limosum together with 60 mM dl-lactate decreased lactate (P < 0.001) and acetate (P < 0.05) and increased levels of E. limosum (P < 0.001) and E. hallii (P < 0.05) compared to 60 mM dl-lactate alone (Compared to 60 mM  < 0.05)  and c. Ate alone .To further demonstrate the impact of treatments on the infant PolyFermS microbiota of donors 1 and 2, we performed principal-coordinate analysis (PCoA) of weighted and unweighted UniFrac distance . In ferm10.1128/mSystems.00264-18.9FIG S9P. avidum; orange, lactate plus E. limosum. Download FIG S9, TIF file, 1.0 MB.PCoA of weighted and unweighted UniFrac distance of treated and nontreated microbiota in control and treatment reactors in fermentations 1 and 2. Red, CR and TRs during no treatment. Other colors, TRs during treatment; purple, pH 5; turquoise, pH 7; yellow, pH 5 plus 10-h RT; pink, 10-h RT; blue, lactate; green, lactate plus Copyright © 2019 Pham et al.2019Pham et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the in vivo studies in humans often face social and ethical challenges due to invasive medical procedures  on lactate production and utilization in batch cultures inoculated with fecal slurries from four adult donors showed that pH 5.2 induced lactate accumulation due to reduced utilization . Using a of life . A recen of 5.97 .Veillonella bacteria, which are the main producers of propionate in the infant gut  and lower abundance of LUB . Consistent with previous studies  increased with prolonged RT  and 17.4 h  at age 17 and 113 days, respectively , while tonged RT . IncreasBifidobacterium and a higher abundance of Enterobacteriaceae relative to a 5-h RT. This observation agrees with previous studies that showed that Bifidobacterium spp. were less abundant in feces from functional constipated adult patients (Enterobacteriaceae levels were higher and Bifidobacterium levels were lower in constipated-irritable bowel syndrome (IBS) adults (C-IBS) compared to healthy adults  may also lead to flatulence and is a possible factor in infantile colic  were shown to compete with high H2-producing LUB  in pure and mixed cultures using anaerobic techniques  was reused, confirming the efficient utilization of lactate by LUB. Furthermore, adding 60 mM dl-lactate to mimic lactate accumulation increased butyrate and propionate formation. Interestingly, the impact of lactate was detected only on a functional but not on a taxonomic level, suggesting that lactate increased the activity of LUB by providing more energetic substrate but not by stimulating growth to detectable levels. Infant LPB, including Lactobacillus, produce both d- and l-lactate. The two isomers of lactate were detected at comparable levels after the addition of 60 mM dl-lactate, suggesting that the 2-month-old infant LUB community was able to utilize both d- and l- forms. Our data suggest that LUB of infant colonic microbiota have a high capacity to metabolize lactate, possibly as a natural protective mechanism in infant microbiota preventing lactate accumulation and detrimental health effects such as acidosis.In this study, a large amount of lactate  was previously isolated from feces of a healthy infant ; 10 g liter−1 yeast extract ; 117 mM sodium dl-lactate 60% syrup ; 0.25 g liter−1 KH2PO4 ; and 5 mg liter−1 MnSO4, 4 mg liter−1 metronidazole, and 10 mg liter−1 kanamycin  in distilled water. Overnight P. avidum cultures  were centrifuged at 7,000 rpm for 10 min, the supernatants were discarded, and the bacterial pellets were washed with 0.1 N sodium phosphate buffer . The resuspended pellets were centrifuged at 7,000 rpm for 10 min and resuspended in sodium phosphate buffer  before being added to the test reactors.y infant . The strE. limosum , previously isolated from feces of a healthy infant  at 37°C under strict anaerobiosis using Hungate tubes flushed with CO2  before being used to inoculate the reactors. The purity of P. avidum and E. limosum cultures was checked via Gram staining.y infant , was actwith CO2 , 49. Twein vitro, both infants selected for this study had been fed exclusively with infant formula , transferred into a gastight anaerobic jar containing a CO2-generating system , and transported at 4°C for processing, immobilization, and reactor inoculation within 3 h of defecation. Immediately upon receipt, the fecal sample was transferred to an anaerobic chamber and immobilized in 1- to 2-mm-diameter gel beads composed of 2.5% (wt/vol) gellan gum, 0.25% (wt/vol) xanthan gum, and 0.2% (wt/vol) sodium citrate as previously described  was collected from diapers, immediately suspended in prereduced peptone water (10 g literescribed .−1): lactose (6.4), casein (0.5), whey protein (8.1), peptone (0.5), Bacto tryptone (0.5), mucin (4), yeast extract (2.5), cysteine (0.8), bile salts (0.05), KH2PO4 (0.5), NaHCO3 (1.5), NaCl (4.5), KCl (4.5), MgSO4·7H2O (1.25), CaCl2·2H2O (0.1), FeSO4·7H2O (0.005), hemin (0.01), Tween 80 (1), and vitamin solution. The vitamin solution contained the following (mg liter−1): pyridoxine-HCl (100), 4-aminobenzoic acid (PABA) (50), nicotinic acid (50), biotin (4), folic acid (4), cyanocobalamin (5), thiamine (50), riboflavin (50), phylloquinone (0.15), menadione (2), and d-pantothenic acid (100). The nutritive medium was freshly prepared daily, autoclaved, and stored at 4°C under stirring until use. All components were from Sigma-Aldrich , except for whey protein , peptone , Bacto tryptone , bile salts , and KH2PO4 .The fermentation medium was based on the composition designed previously to mimic the chyme entering the colon of 6-month-old infants , 50. The2. Temperature was set at 37°C, stirring speed was set at 180 rpm, and pH was maintained automatically at 6.0 by adding 2.5 M NaOH.The PolyFermS continuous fermentation model used in this study was designed to mimic conditions in the proximal colon of a 2-month-old formula-fed infant. The fermentation setup consisted of a first reactor with a working volume of 200 ml inoculated with 60 ml  fecal beads from the respective donor (IR), which was connected to a control reactor (CR) and four test reactors (TRs) . All TRs−1, corresponding to a mean RT of 5 h. This flow rate simulated the transit time in the infant proximal colon, which is estimated to be a total transit time of 17.4 h in formula-fed infants aged 113 days  to colonize beads in the IR. During colonization (days 1 and 2), fermentation effluent was replaced by fresh medium every 12 h . Afterwa113 days . After adl-lactate was added in all TRs to achieve a concentration of 60 mM, with or without daily addition of E. limosum (108 CFU ml−1) and P. avidum (1 × 108 or 5 × 108 CFU ml−1).The IR and CR were operated with constant conditions of pH 6.0 and 5-h RT throughout the fermentation time, which was 79 and 57 days for F1 and F2, respectively. Detailed experimental conditions for the two PolyFermS fermentations are depicted in P. avidum were performed in triplicate on samples collected during the last 3 days of stabilization, P. avidum treatment, and posttreatment periods (F2).Sampling of effluents from all reactors was performed daily. The sample supernatant  was used for HPLC analysis, while the pellet was stored at −80°C for DNA extraction. HPLC and qPCR were performed on samples collected during the last 3 days of each stabilization and treatment. MiSeq sequencing was performed on pooled samples collected during the last 2 days of the periods. Plate counts of Total genomic DNA was extracted from 200 mg fresh infant feces and the pellet from 2 ml of fermentation effluent samples using the FastDNA Spin kit for soil  according to the manufacturer’s instructions. DNA concentration and quality were assessed by absorbance measurements at 260 nm on a NanoDrop ND-1000 spectrophotometer , and samples were stored at −20°C before qPCR and MiSeq sequencing analyses.qPCR was performed using an ABI Prism 7500 PCR sequence detection system . Specific primers targeting predominant bacterial groups or species in the infant gut were used at a final concentration of 0.2 μM see .de novo operational taxonomic units (OTU) were conducted using the UPARSE pipeline. The HIT 16S rRNA gene collection was used as a reference database.V3-V4 amplicons were prepared using specific forward primer F340 (5′-CCTACGGRAGGCAGCAG-3′) and reverse primer R805 (5′-GGACTACHVGGGTWTCTAAT-3′). Illumina MiSeq sequencing analyses of fecal and effluent samples were carried out at Genotoul . Thermocycling was performed with an initial step at 94°C for 60 s, followed by 30 cycles of denaturation at 94°C for 60 s, annealing at 65°C for 60 s, and elongation at 72°C for 60 s, with a final elongation of 10 min at 72°C. The raw data set containing paired-end reads with corresponding quality scores was merged and trimmed using settings as previously mentioned . QuantitPropionibacterium amplification by qPCR, P. avidum was enumerated in duplicate by plating 100 μl of effluent sample, which had been serially diluted 10-fold, on 1.5% sodium lactate agar supplemented with metronidazole (4 mg liter−1) and kanamycin (10 mg liter−1)   and some Gram-positive bacteria  . Antibiorganisms , such as species , and kanbacteria , 56. A cus sizes . Plates dl-lactate in effluent samples from all reactors were determined by HPLC analysis. Supernatants from effluent samples were passed through 0.45-μm nylon HPLC filters  before injection. HPLC analysis  was performed as described previously (−1 effluent (mM).The concentrations of SCFAs , formate, and l-Lactate concentration was measured by an enzymatic kit according to the manufacturer’s instructions . d-Lactate concentration was determined by subtracting l-lactate concentration from total dl-lactate concentration.10-transformed) and HPLC data were expressed as the mean results ± SD for the last 3 days of each fermentation period and compared pairwise between stabilization and treatment within each TR, using repeated-measures ANOVA. Comparisons between reactors within each fermentation period were performed using ANOVA after testing for normal distribution using the Shapiro-Wilk test.Statistical analysis was done using IBM SPSS Statistics 20.0 . qPCR  between treatment and stabilization period within each reactor were calculated for each combination of 3 measurement days, resulting in 9 delta values per fermentation. Delta values between treatment (TR1 to TR4) and control (CR) reactors were compared using the Wilcoxon rank sum test with false-discovery rate correction. Pairwise comparisons of SCFA concentrations and bacterial population levels between each treatment reactor (TR1 to TR4) and control reactor (CR) during stabilization periods were carried out using the Wilcoxon rank sum test with false-discovery rate correction. For all tests, PRJEB32244).The sequence data reported in this paper have been deposited in the European Nucleotide Archive database (accession no.


Blood samples were taken before and after the class. The dance class had no effect on the plasma concentration of CRP, TNF-α, IL-6, IL-10, and IL-8; however, we noted an increase in levels of IL-1β (4.06%) and sL-selectin (17.67%). The dance class resulted in a 12.36% increase in neutrophil counts, while neutrophil CD62L expression and migration were reduced . After the dance class, neutrophil production of IL-8 and TNF-α increased, respectively, by 59.75% and 49.23%, in the control condition, and 43.55% and 32.22%, after LPS stimulation. A single bout of street dancing induced inflammation and reduced neutrophil migration and adhesion molecule expression. These findings may contribute to a better understanding of the susceptibility to infection after acute dance exercise.The physical demands of street dancing may result in inflammation and changes in leukocyte numbers/function, impairing the health of dancers. Herein, we investigated the effect of street dancing on inflammation, adhesion molecules, and neutrophil function. Fifteen amateur dancers (mean ± SE: age 22.4 ± 1.08 years, BMI 24.8 ± 0.69 kg/m Hip-hop dance, a term that encompasses many styles, including popping, breakdance, funk, house, krumping, voguing, and street dance , has groα (TNF-α) and interleukin 1β (IL-1β)) into the injured tissue [Under normal conditions, after muscle damage is induced, the inflammatory response is initiated by the release of inflammatory mediators, such as proinflammatory cytokines , interleukin-10 (IL-10), and interleukin-8 (IL-8)) and C-reactive protein (CRP) measurement. We also assessed sL-selectin levels, neutrophil numbers, migration, expression of CD62L and IL-8, and TNF-α release by stimulated and nonstimulated neutrophils.In order to understand inflammatory modulation by neutrophils, studies have used treatment with lipopolysaccharide (LPS) stimulus to induce changes in Toll-like receptor 4 signaling pathways and NF-kappa B activation, simulating the exaggerated host response to infection through the release of cytokines and adhesion molecules . Moreove2, fat-free mass 61.5 ± 1.64 kg, body fat 12.3 ± 1.52%, and dance experience of 7 ± 13.78 years. The dancers had a training/performance history of 3.7 ± 0.39 hours a day, four days a week, and rested on weekends.With the approval of the Ethics Committee at the Cruzeiro do Sul University (0522013), 15 male dancers volunteered to participate in the study. All dancers signed an informed consent form agreeing to submit to the procedures involved in the study. The group had the following characteristics (mean ± standard error (SE)): age 22.4 ± 1.08 years, weight 70.8 ± 1.93 kg, height 169 ± 0.02 cm, body mass index (BMI) 24.8 ± 0.69 kg/m3/mm3, white blood cells (WBC) 9 ± 0.53 × 103/mm3, hemoglobin (Hb) 14.7 ± 0.29 g/dL, hematocrit (Ht) 44.2 ± 0.88%, mean red cell volume 83 ± 1.86 fL, and hemoglobin concentration per red blood cell (MCHC) 33.3 ± 2.40%.Participants were excluded from the study if they had a history of recent infection (last 15 days prior to the study), viruses, chronic lesions, diabetes, rheumatoid arthritis, hormonal dysfunction, lupus, or other inflammatory and hematology diseases (such as hemoglobinopathies) and took medication. To verify the dancer's health level, hematological parameters were assessed. The group presented the following characteristics (mean ± SE): red blood cells (RBC) 5.4 ± 0.14 × 10Polar HR monitors and danced for 60 minutes at high intensity. This was defined according to the following intensities via HR reserve: at 85 ± 5.9% in the first 20 minutes, at 88.9 ± 3.9% between 20 and 40 minutes, and at 89.2 ± 3.3% between 40 and 60 minutes of the class.For this study, dancers were asked to rest for 72 hours before the street dance class. During the class, all participants had their heart rate (HR) monitored using Twenty milliliters (mL) of venous blood was collected before and immediately after the street dance class. Blood samples were drawn into BD Vacutainer® tubes, containing heparin. After collecting the samples, blood was centrifuged at 400 ×g for 10 minutes and the plasma was used to evaluate cytokines and CRP. Moreover, neutrophils were isolated from whole blood for cellular analyzes.α, IL-6, IL-10, IL-8, sL-selectin, and IL-1β) were measured by ELISA, according to the manufacturer's instructions . A standard curve was produced for each set of samples, and the cytokine was assayed [Plasma CRP and cytokines (TNF- assayed .Cell isolation was carried out within 1 hour of venipuncture . Human n6. Neutrophils (2.5 × 106) were suspended in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 0.3 g/L glutamine, 2.32 g/L Hepes, 2 g/L sodium bicarbonate, 100 μg/mL streptomycin, 100 UI/mL penicillin, and 10% fetal bovine serum. The cells were then counted in a Neubauer chamber and immediately cultured at 37°C and 5% CO2, with and without LPS (5 μg/mL) supplied by Sigma Chemical Co. . After 18 hours of cell culture, the supernatant was collected and stored at ≤−80°C prior to cytokine analysis (IL-8 and TNF-α) by ELISA .After isolation, the cultured cell concentration obtained was 87.6 × 106 cells/mL). In summary, the MEL-14 monoclonal antibody is conjugated with the fluorochrome fluorescein isothiocyanate (FITC) and recognizes an epitope localized to the lectin domain and CD62L binds a number of fucosylated, glycosylated, sulfated sialylated glycoproteins including CD34, MAdCam-1, and glycam-1. Data were acquired and analyzed using Diva software (Becton Dickinson), and results were expressed as the mean of the FITC fluorescence intensity. Ten thousand events were analyzed per experiment.The expression of CD62L  was measured with a FACSAria II flow cytometer  from neutrophils (2.5 × 10μm were fixed to the filter seat. The chamber assembly with neutrophils (2.5 × 104 cells/mL) was incubated in a humidified 5% CO2 atmosphere at 37°C for 60 minutes. After incubation, the chamber was disassembled and the cells that migrated, crossing the plasma membrane, were counted directly in the Neubauer chamber and chemotactic responses were defined as the mean number of migrated neutrophils. The chemotactic factor fMLP (10 nmol/L) was used as a positive control for migration. Results were normalized to the number of cells that transmigrated under control conditions (PBS without fMLP).Neutrophil chemotactic responses were measured using 96-well disposable chemotactic plates (Neuro Probe), according to the manufacturer's instructions. Briefly, flat-bottomed chambers were filled with the chemotactic agent N-formyl-methionyl-leucyl-phenylalanine (fMLP)  (10 nmol/L) in phosphate-buffered saline (PBS) containing 0.01% albumin. Chemotactic membranes with a pore size of 5 t-test. Analysis of cytokine production by neutrophils (IL-8 and TNF-α) was performed using a one-way analysis of variance (ANOVA) accompanied by post hoc test (Student-Newman-Keuls multiple comparison) . Significance was accepted at p < 0.05. Linear relationships were explored by Pearson's correlation.The results are expressed as the means ± SE. Statistical analysis of plasma cytokines and CRP, neutrophil counts, cell migration, and CD62L expression data was performed by paired α, IL-6, IL-10, and IL-8   .p < 0.05) and in the concentration of sL-selectin   and neutrophil migration   and TNF-α (45.9% (baseline) and 27.77 (post), p < 0.05)  and 49.23% (p < 0.01), respectively, in the basal condition, and by 43.55% (p < 0.05) and 32.22% (p < 0.05), respectively, in LPS-stimulated conditions, after the street dance class.LPS stimulation resulted in an increase in neutrophil production of IL-8 (41.95% (baseline) and 18.59 (post),  Figures , resp..  Figures  and TNF-igures α  increaser2 = 0.60, p < 0.0001)  ( 0.0001) , and a n < 0.05) .β and sL-selectin, indicating the induction of inflammation. sL-selectin, from neutrophils, is found in the plasma at high levels, and it may retain functional activity, indicating a possible role for sL-selectin in the regulation of leukocyte attachment to endothelium [Our results demonstrated that a single session of street dancing, lasting 60 minutes, increased the concentration of IL-1othelium . sL-seleothelium . Interesothelium , our datα in the control and LPS-stimulated conditions were increased and there was a reduction in neutrophil migration immediately after the dance class. In contrast to our data, some investigations have reported increased neutrophil chemotactic activity after moderate exercise [2 max). Similar to our data, they found that neutrophil chemotaxis was impaired after exercise [After the class, neutrophil count increased but the expression of CD62L on neutrophils was lower. In addition, we found that neutrophil productions of IL-8 and TNF-exercise , 16. Howexercise , 18, andexercise . In ordeexercise  studied exercise . They suexercise , a hypotexercise , 22.Although the clinical significance of the transient reduction in neutrophil chemotaxis after intense exercise, such as street dance, is still not clear, it is important to keep in mind that neutrophil function is a key component in the resolution of infection and the control of inflammation . Once in2 max for 30 minutes every day). Plasma concentrations of TNF-α and soluble Fas ligand were raised during the exercise sessions and had not returned to normal 72 hours after the completion of exercise [In fact, short-term, high-intensity exercise can lead to a significant and prolonged dysfunction of the peripheral blood leukocytes, which is accompanied by an increase in proinflammatory mediators. To illustrate this, Tuan et al.  investigexercise . This suexercise .The current study found that neutrophil numbers increased immediately following a street dance class. However, the expression of CD62L by neutrophils was reduced by the class. We observed a negative correlation between exercise intensity and expression of CD62L, indicating that effect of exercise on the expression of adhesion molecules, an important event for the rolling, adhesion, and migration of neutrophils , is inteα was higher with LPS stimulation. An important aspect of neutrophil function is their ability to modulate the inflammatory response via the systemic production of cytokines, such as TNF-α and other inflammatory mediators [α and IL-8 in the nonstimulated (absence of stimuli) and stimulated conditions. This increased production of cytokines by neutrophils after the dance class points to dancing as a priming agent for subclinical inflammation.As expected, the neutrophil production of IL-8 and TNF-ediators . These lediators . Moreoveα, IL-1β, and IL-8 are associated with the severity of inflammatory processes [β in the current study agrees with the current literature, as other studies have shown that intense exercise [The serum concentrations of TNF-rocesses . Also, texercise , and eveexercise , inducesexercise , 28. Morexercise  demonstrexercise ; howeverexercise , 31. TheIntensive exercise suppresses several leukocyte functions including chemotaxis and the release of cytokines by leukocytes . The finAn acute bout of street dancing induces inflammation and reduces neutrophil migration and adhesion molecule expression. These findings improve our understanding of the risk of infection following acute dance exercise, and it may represent a useful tool to design new studies and strategies to prevent a dancer's performance from declining as a result of inflammation/infection.


SoGloI was identified from sugarcane. SoGloI had a size of 1,091 bp with one open reading frame (ORF) of 885 bp encoding a protein of 294 amino acids. SoGloI was predicted as a Ni2+-dependent GLOI protein with two typical glyoxalase domains at positions 28–149 and 159–283, respectively. SoGloI was cloned into an expression plasmid vector, and the Trx-His-S-tag SoGloI protein produced in Escherichia coli was about 51 kDa. The recombinant E. coli cells expressing SoGloI compared to the control grew faster and tolerated higher concentrations of NaCl, CuCl2, CdCl2, or ZnSO4. SoGloI ubiquitously expressed in various sugarcane tissues. The expression was up-regulated under the treatments of NaCl, CuCl2, CdCl2, ZnSO4 and abscisic acid (ABA), or under simulated biotic stress conditions upon exposure to salicylic acid (SA) and methyl jasmonate (MeJA). SoGloI activity steadily increased when sugarcane was subjected to NaCl, CuCl2, CdCl2, or ZnSO4 treatments. Sub-cellular observations indicated that the SoGloI protein was located in both cytosol and nucleus. These results suggest that the SoGloI gene may play an important role in sugarcane’s response to various biotic and abiotic stresses.Glyoxalase I belongs to the glyoxalase system that detoxifies methylglyoxal (MG), a cytotoxic by-product produced mainly from triose phosphates. The concentration of MG increases rapidly under stress conditions. In this study, a novel glyoxalase I gene, designated as  Ubiquitously occurring in nature, the glyoxalase pathway involves a two-step catalytic reaction. In the first step, glyoxalase I  catalyzes the isomerization of hemithioacetal formed spontaneously between methylglyoxal (MG) and reduces glutathione (GSH) to S-D-lactoylglutathione (S-LG) . In the 2+-or Ni2+-dependent class of metal activation. Previous studies have been showed that the Zn2+-dependent GLOI enzymes are thought to be of eukaryotic origin  is a cash crop mainly used for sugar, biofuel and other food industries such as industrial alcohol in tropical and subtropical regions. It is one of the world’s largest crops. According to the FAO, sugarcane was cultivated in 101 countries on about 26.1 million hectares of land in 2012 (Sugarcane ( in 2012 . However in 2012 . Though  in 2012 . RSD cau in 2012 . Sugarca in 2012 .Sporisorium scitamineum-infected buds (CA140600.1) had a high similarity to the GLOI gene of Zea mays (GenBank Accession Number: EU966885.1)  . To studSugarcane genotype YCE 05-179 was used in this study. Plants were maintained in a genetic nursery at the Key Laboratory of Sugarcane Biology and Genetic Breeding, Ministry of Agriculture, Fujian Agriculture and Forestry University, Fuzhou, China. In addition, tissue culture-derived young, healthy plantlets of YCE 05-179 were also involved in the study.http://www.ncbi.nlm.nih.gov/) was conducted with the sugarcane EST, CA140600.1 . SoGloI cDNA sequence was amplified in a 25 µL reaction mixture on ABI Veriti96 PCR with primers G-F and G-R (2+), 2.5 µL dNTPs (2.5 mM), 1.0 µL first-strand cDNA, 1.0 µL each of forward and reverse primers (10 µM) and 0.125 µL Ex-Taq enzyme (5 U µL−1) . Amplified SoGloI cDNA product was separated through 1% agarose gel electrophoresis, gel purified using Omega Gel Extraction Kit , and cloned into the PMD 18-T vector . Putative recombinants were confirmed by PCR, of which six were sequenced .A BLAST search of the sugarcane database  of GenBank . Molecular weight and isoelectric point was calculated using Compute pI/Mw tool (http://www.expasy.ch/tools/pi_tool.html). Protein domain was predicted using the SMART Program (http://smart.embl-heidelberg.de/). Protein hydrophilicity was analyzed by Protscale Program . Prediction of signal peptides and analysis of trans-membrane helix domain were conducted using the SignalP and TMHMM-2.0 Programs (http://www.cbs.dtu.dk/services/). A multiple protein-sequence alignment was carried out using the DNAMAN® Program  according to the manufacturer’s instructions. RNA samples were dissolved in diethylpyrocarbonate-treated H2O. RNA concentration was quantified by measuring absorbance at OD260 and OD280 using Synergy H1 Microplate Reader Multi-Mode . RNA quality was also assessed by 1.0% denaturing agarose gel electrophoreses.Five tissue samples, white young roots, leaf (+1), leaf sheath (+1), buds (6th-8th from the base), and internodes (6th and 7th from the base) were collected from 7- to 8-month-old plants in the field nursery. All samples except buds were cut into small pieces, wrapped up within tinfoil, and immediately flash-frozen in liquid nitrogen. The frozen samples were stored in a −80 °C freezer until RNA extraction. Total RNA samples were isolated from the frozen buds using the TRIzol® 1st Strand cDNA Synthesis Kit  on ABI Veriti96 PCR . For real-time quantitative PCR (RT-qPCR), a total of 1 µg RNA in 20 µL reaction mixtures was used for the first-strand cDNA synthesis using the Prime-Script® RT Reagent Perfect Real Time Kit  on ABI Veriti96 PCR. Phosphoglyceraldehyde dehydrogenase (GAPDH) (CA254672) gene was used as the internal control. The primers of GAPDH-QF/GAPDH-QR were listed in First-strand cDNA was synthesized from 1 µg RNA in 20 µL reaction mixture by reverse transcription PCR (RT-PCR) using the Prime-Script2; 100 µM CuCl2; or 100 µM ZnSO4 for 12, 24 and 48 h  plastic trays, containing only distilled water. The water was changed every morning. After 10 days, the plantlets were transplanted to 100-mL flat-bottomed glass tubes and subjected to seven different stress treatments in three replicates. Three plantlets of each treatment was treated with the following solutions: 100 µM methyl jasmonate  ethanol and 0.05% (v/v) Tween-20); 5.0 mM salicylic acid  Tween-20); 100 µM abscisic acid (ABA) for 6, 12, and 24 h ; 250 mM and 48 h , respectSoGloI expression in response to MeJA, SA, ABA  and NaCl, CdCl2, CuCl2, ZnSO4  was analyzed by RT-qPCR on a 7500 RT-qPCR system . 25S rRNA (BQ536525) gene was chosen as internal control and. The primers of 25S-QF/25S-QR were listed in SoGloI-specific primer pair of G-QF/G-QR was designed by Primer Premier 5.0 software  . The reactions were repeated three times for each sample. The 2−ddCt method was used to calculate relative gene expression levels  . RT-qPCRn levels .SoGloI gene fragment was amplified from the cDNA clone with primers G-32aF/G-32aR  and subsequently sub-cloned into the EcoR I–Xho I sites of pET32a (+)  . Expression of pET32a-SoGloI was induced in 1 mM isopropyl β-D-thiogalactoside (IPTG) for 8 h at 37 °C and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) at 0, 2, 4, and 8 h. Non-transformed (blank) and the vector pET32a-transformed Rosetta cells (control) were used as the controls.A F/G-32aR . The SoGT32a (+)  to produSoGloI transformed E. coli cells to NaCl, CdCl2, CuCl2 or ZnSO4 treatments. When the E. coli culture mixture reached OD600 = 0.6, 1 mM IPTG was added into the LB medium and the culture mixture was incubated for 12 h at 28 °C. Then the cultures were first diluted to 0.6 (OD600), and further diluted to two levels (10−3 and 10−4)  . Thereafectively . All pla2 in a mortar. Protein was extracted with an extraction buffer containing 0.1 M potassium phosphate buffer , 50% (v/v) glycerol, 16 mM MgSO4, 0.2 mM Phenylmethanesulfonyl fluoride (PMSF) and 0.2% (v/v) polyvinylpyrrolidone (PVP40). The extract was centrifuged twice at 13,000 rpm at 4 °C for 30 min to obtain the crude protein extract from the supernatant (−1 cm−1.Entire flash-frozen 4-month old plantlets (100 mg wet weight) were pulverized in liquid Nernatant . The supernatant  using boSoGloI gene was sub-cloned into the Xcm I/BamH I restriction sites of pCXSN to construct a fusion protein expression vector of 35S::SoGloI::GFP. The GFP-containing pCXSN vector was a gift of Songbiao Chen, Institute of Biotechnology, Fujian Academy of Agricultural Sciences. The pCXSN-SoGloI recombinant plasmids were transformed into Agrobacterium tumefaciens cells, strain GV 3101 (−1) and rifampicin (34 µg mL−1). The culture was incubated overnight at 28 °C with shaking at 200 rpm. The culture was then centrifuged at 5,000× g to harvest the Agrobacterium cells followed by, re-suspension in 10 mM MgCl2 and 10 mM fatty acid methyl ester sulfonate (MES). The concentration of the bacterial suspension was measured and adjusted to OD600 = 0.6 with Murashige and Skoog (MS) liquid medium supplemented with 200 mM acetosyringone. The resulting culture was incubated at 28 °C for 3 h  supplemented with 0.4 M sucrose, 1.5% cellulase R-10  and 0.3% macerozyme R-10 (Yakult). The leaf tissue was vacuum-infiltrated for 30 min at 20 mm Hg and digested at room temperature with gentle shaking for 4 h to produce protoplasts. Then, the K3 medium was replaced with 12 mL of W5 solution . The protoplasts were collected by centrifugation at 300× g for 4 min at 4 °C and re-suspended in 1 mL WI solution . The sub-cellular location of the SoGloI gene was observed using fluorescence microscopy .The  GV 3101 . The tra for 3 h . Then, 1 for 3 h . Then, tSoGloI (GenBank Accession Number: KC857628) was 1,091 bp with one ORF of 885 bp , Arabidopsis thaliana , Pseudomonas aeruginosa , Glycine max (G. Zn-GloI and G. Ni-GloI) and Escherichia coli (GlxI) was done using DNAMAN®   , Row b. o 250 mM  as well o 250 mM –3E. Cons µM ZnSO4. These rSoGloI. The SoGloI gene was ubiquitously expressed in five tissues of 7- to 8-month old plants collected from the field. The highest level was detected in buds, followed by leaves, roots, leaf sheaths, and internodes , and GloA3 (Zn binding). Arabidopsis thaliana. AtGLYI2 was found to be Zn2+-dependent, whereas AtGLYI3 and AtGLYI6 were Ni2+-dependent. Ni2+-dependent GloI is present as a two-domain protein in all eukaryotes. Among the early branching eukaryotes, the group of algae appears to be the first to encode this gene , a micronutrient, is necessary for plant growth, but an excessive amount of Zn2+ can inhibit plant growth  showed that GlyI from B. juncea enhanced host Zn2+ tolerance to toxic levels in the transgenic tobacco. The expression of TaGly I, a glyoxalase I gene of T. aestivum, is induced continuously under 20 mM ZnCl2treatment. Compared to control, the increase in TaGly I expression is nearly 1.5-fold at 24 h  with a shorter life cycle and simpler genome can be explored as an alternative host for transforming and assessing the functional properties of isolated sugarcane genes, such as SoGloI.As is known, sugarcane is a polyploidy and aneuploidy crop , in whicSoGloI) gene in sugarcane. We isolated and characterized SoGloI gene and demonstrated the enzyme activity of glyoxalase I protein. We found that SoGloI expression and SoGloI enzymatic activity were elevated significantly when sugarcane tissues were subject to simulated biotic and abiotic stress conditions, such as high concentrations of salt or heavy metal ions. The findings have opened up a new research avenue for sugarcane to grow in polluted or salty environments via genetic engineering and breeding of SoGloI to enhance host resistance.This is the first report on the cloning and characterization of glyoxalase I (10.7717/peerj.5873/supp-1Figure S1Click here for additional data file.10.7717/peerj.5873/supp-2Figure S2Click here for additional data file.10.7717/peerj.5873/supp-3Figure S3Click here for additional data file.10.7717/peerj.5873/supp-4Figure S4Click here for additional data file.10.7717/peerj.5873/supp-5Figure S5Click here for additional data file.10.7717/peerj.5873/supp-6Data S1Click here for additional data file.


We evaluated the safety and efficacy of induction chemotherapy with bevacizumab followed by maintenance chemotherapy with bevacizumab for advanced non-small cell lung cancer (NSCLC) in this multicenter phase II study.2), pemetrexed (500 mg/m2), and bevacizumab (15 mg/kg) followed by maintenance with pemetrexed (500 mg/m2) and bevacizumab (15 mg/kg) until disease progression. Progression-free survival (PFS) was the primary endpoint.Chemotherapy-naïve patient with stage IIIB–IV or recurrent nonsquamous NSCLC were eligible. We planned approximately four cycles of induction cisplatin  patients received a median of nine maintenance chemotherapy cycles. The objective response was 70.6%, and the disease control rate was 97.1%. The median PFS was 10.8 , and overall survival was 48.0  months. Median PFS of 23 patients with epidermal growth factor receptor (EGFR) mutations and of 16 patients without EGFR mutations were 12.9  and 7.9  months, respectively. Toxicities graded ≥3 included neutropenia (15%), anemia (15%), hypertension (7.5%), anorexia (7.5%), fatigue (7.5%), thromboembolic events (5%), jaw osteonecrosis (5%), nausea (2.5%), oral mucositis (2.5%), tumor pain (2.5%), hyponatremia (2.5%), and gastrointestinal perforation (2.5%). Treatment-related deaths were not found.In patients with advanced or recurrent nonsquamous NSCLC, induction chemotherapy with cisplatin, pemetrexed, and bevacizumab followed by maintenance chemotherapy with pemetrexed and bevacizumab is safe and effective regardless of their EGFR mutation status.UMIN000005569. Registered date: May 8, 2011.UMIN Clinical Trial Registry:  Table n = 1), unacceptable toxicity (n = 3), and patient request (n = 1).The 40 eligible patients were administered a median of four cycles  of induction chemotherapy comprising cisplatin, pemetrexed, and bevacizumab. Of these patients, 35 (87.5%) were administered maintenance chemotherapy comprising pemetrexed and bevacizumab for a median of nine cycles  Table . Reasons> 3 included 15.0% with neutropenia, 15.0% with anemia, 7.5% with hypertension, 7.5% with anorexia, 7.5% with fatigue, 5% with a thromboembolic event, 5% with jaw osteonecrosis, 2.5% with nausea, 2.5% with oral mucositis, 2.5% with tumor pain, 2.5% with hyponatremia, and 2.5% with gastrointestinal perforation. There were no treatment-related deaths.Table n = 34) , and OS was 48.0  months  and those without the mutation (n = 16) were 12.9  and 7.9  months, respectively  had a median OS of 20.7  months , pemetrexed (500 mg/m2), and bevacizumab (15 mg/kg) followed by maintenance therapy with pemetrexed (500 mg/m2) and bevacizumab (15 mg/kg) in patients with advanced or recurrent nonsquamous NSCLC. Non-hematological toxicities graded higher than 3 were not found in 10% of the patients, and a grade 4 non-hematological toxicity  developed in only one (2.5%) patient. Furthermore, there was no treatment-related death in this study. The low frequencies of severe toxicities observed in this study are consistent with those observed in other studies [We demonstrated the safety and efficacy of induction therapy using cisplatin 75 mg/m5 mg/m2,  studies . Serious studies . To the Regarding efficacy, this study met its primary endpoint with a median PFS of 10.8 months, which is consistent with the results of the phase III AVAPERL study  , and seeThis study included 57.5% patients with EGFR mutations. In our EGFR status-based sub-analysis, PFS of patients with EGFR mutations seemed longer  than that of those without the mutations ; however, this difference was not significant. PFS of patients with EGFR mutations who received cisplatin, pemetrexed, and bevacizumab in the present study was similar to that of those who received EGFR-TKIs in other studies  . The sta2), pemetrexed (500 mg/m2), and bevacizumab (15 mg/kg) followed by maintenance with pemetrexed (500 mg/m2) and bevacizumab (15 mg/kg) is safe and effective for patients with stage III, IV, or recurrent nonsquamous NSCLC, and even for those with EGFR mutations.A first-line combination therapy with cisplatin (75 mg/m


O-Methylated phenylpropanoids, which are generally present in small amounts in plants, have improved or distinct biological activities and pharmacological properties as opposed to their unmethylated counterparts. Although microbial production could be a useful tool for the efficient and environment-friendly production of methylated phenylpropanoids, a high-yield microbial production of neither tri-methylated stilbenes nor di-/tri-methylated flavonoids has been achieved to date.Streptomyces avermitilis (SaOMT2), which has been known to possess 7-O-methylation activity toward several flavonoids, exhibited more diverse regiospecificity and catalyzed mono-, di-, and tri-methylation of stilbene, flavanone, and flavone when it was expressed in Streptomyces venezuelae. For the efficient production of multi-methylated phenylpropanoids, a cocultivation system was developed by employing engineered Escherichia coli strains producing pterostilbene, naringenin, and apigenin, respectively, along with SaOMT2-expressing S. venezuelae mutant. Consequently, high-yield microbial production of tri-methylated stilbenes and di-/tri-methylated flavonoids  has been demonstrated for the first time.A methyltransferase from E. coli and SaOMT2-expressing S. venezuelae provides an efficient tool for producing scarce and potentially valuable multi-methylated phenylpropanoids and will enable further development of these compounds as pharmaceuticals and nutraceuticals.This cocultivation system based on the phenylpropanoid-producing The online version of this article (10.1186/s12934-019-1118-9) contains supplementary material, which is available to authorized users. Phenylpropanoids, including flavonoids and stilbenes, are structurally diverse plant secondary metabolites that have significant potential as pharmaceuticals, nutraceuticals, and cosmetics owing to their antioxidation, cancer prevention, anticancer, antibacterial, and anti-inflammatory properties . HoweverPyricularia oryzae, but only 10 mg of sakuranetin can be isolated from 50 g of ultraviolet-irradiated rice leaves [Aglaia duperreana and Praxelis clematidea R.M. King & Robinson; this naringenin trimethyl ether exhibited considerable molluscicidal activity against golden apple snail, an agricultural pest [Candida species [Saccharomyces cerevisiae by expressing resveratrol biosynthetic genes and O-methyltransferase (OMT) gene from Vitis vinifera (VvROMT) and carrying out further optimization [Oryza sativa L. cv. Nakdong in Escherichia coli produced approximately 40 mg/L of sakuranetin  [Sorghum bicolor in E. coli [It has been reported that methylated flavonoids and stilbenes exhibit significantly higher metabolic stability, oral bioavailability, and biological activity than their unmethylated counterparts . For exae leaves . Tri-mete leaves , has beee leaves , 10. Furral pest , antibacral pest , and ant species . Fortunamization . Express (POMT7) . However E. coli . NotablyStreptomyces avermitilis ATCC 31267 (SaOMT2). SaOMT2 expressed in E. coli reportedly exhibits 7-O-methylation activity toward naringenin as well as some isoflavones and flavones [Streptomyces venezuelae was able to di- and tri-methylate resveratrol, naringenin, and apigenin. Furthermore, we developed a cocultivation system employing each of the engineered E. coli strains that produced pterostilbene, naringenin, and apigenin, respectively, along with SaOMT2-expressing S. venezuelae mutant for achieving a high-yield production of multi-methylated stilbene, flavanone, and flavone. Optimization of cocultivation conditions resulted in the production of approximately 29 mg/L of 3,5,4′-trimethoxystilbene, which is the highest titer reported to date. To our knowledge, the production of a series of di- or tri-methylated naringenin and apigenin analogs based on a cocultivation system is the first report concerning microbial production of multi-methylated flavonoid compounds. This one-pot cocultivation system provides a facile tool for the high-yield production of such scarce and potentially valuable methylated flavonoids and stilbenes and will enable further development of these compounds.Here, we recharacterized the function of 7-OMT from flavones . HoweverE. coli strain expressing one of the OMT from S. avermitilis ATCC 31267  can catalyze 7-O-methylation of flavanone (naringenin), isoflavone (daidzein and genistein), and flavone  [Streptomyces species, we opted to re-examine the regiospecificity of SaOMT2 expressed in a Streptomyces host. In our previous study, S. venezuelae DHS2001, in which the native pikromycin polyketide synthase gene was deleted, has been successfully used to produce flavanones and stilbenes by heterologous expression of codon-optimized genes derived from various plants [S. venezuelae DHS2001 using a replicative E. coli–Streptomyces shuttle vector pSE34 [ermE* promoter [S. avermitilis was expressed in the Streptomyces heterologous host, SaOMT2 not only shows mono-methylation activity on 7-hydroxy group of flavanone, isoflavone, and flavone but also di- and tri-methylation activity on the positions 3, 5, and 4′ of stilbene and the positions 5, 7, and 4′ of flavanone/flavone.It has been reported that the amnetin) . Neverths plants . To conspromoter . ResveraS. venezuelae and E. coli and subjected it to purification  as a methyl donor, the products corresponding to genkwanin, 5-hydroxy-7,4′-dimethoxyflavone, and 5,7,4′-trimethoxyflavone were produced at conversion yields of approximately 26%, 10%, and 0.1%, respectively  is known to efficiently produce pterostilbene from resveratrol [E. coli strains were also constructed. The genes for the synthesis of phenylpropanoid backbones were synthesized but were not codon-optimized because it had been reported that the plant genes could be successfully used for the production of desired compounds in E. coli [O. sativa  [V. vinifera  [V. vinifera (VvROMT)  [Medicago truncatula  [S. venezuelae, which is insensitive to ampicillin. The pAPG plasmid was designed for biosynthesizing apigenin by adding the synthetic FNS gene from Petroselinum crispum to pNRG  [E. coli BL21(DE3) to generate BL21/PTS, BL21/NRG, and BL21/APG, respectively , were grown on LB medium for 3 h at 37 °C and subsequently transferred to R2YE medium [Streptomyces. UPLC–qTOF–HR-MS analysis of organic extracts obtained from the engineered E. coli strains revealed peaks that were consistent with those of authentic pterostilbene, naringenin, and apigenin standards  ; and OMTTC29789) , and CHIY230247) . These eStreptomyces venezuelae has a relatively faster growth rate (an approximate doubling time of 1 h) than that of other streptomycetes including Streptomyces coelicolor and Streptomyces lividans, which are widely-used as heterologous hosts [E. coli, the precultivation period for phenylpropanoid-producing E. coli and SaOMT2-expressing S. venezuelae should be adjusted. We first decided to mix 3 h-old E. coli after induction and 40 h-old DHS2001/SaOMT2 (when it shows maximum cell growth) at the beginning of cocultivation. Next, to optimize the mixing ratio for E. coli and S. venezuelae mutants, the E. coli cells of BL21/PTS, BL21/NRG, or BL21/APG that were recovered by centrifugation were mixed with the recovered S. venezuelae cells of DHS2001/SaOMT2 grown on R2YE medium at varying weight ratios of 1:1, 1:3, 1:6, and 1:9 to make up a total volume of 15 mL in R2YE medium. The cocultures were fed with 1.2 mM of 4-coumaric acid and further incubated for 40 h at 30 °C. The production of 3,5,4′-trimethoxystilbene, which had undergone an additional O-methylation at the position 4′ of pterostilbene, from the coculture of BL21/PTS and DHS2001/SaOMT2 was analyzed by UPLC–qTOF–HR-MS. A cocultivation system in which E. coli and S. venezuelae were mixed at a ratio of 1:3 presented the highest yield of 3,5,4′-trimethoxystilbene (~ 29 mg/L)  as well as mono-methylation at the position 7 of the same compound  not only catalyzes 7,4′- and 5,7-di-methylation but also 5,7,4′-tri-methylation of flavanone (naringenin) and flavone (apigenin) by carrying out whole-cell bioconversion. Additionally, DHS2001/SaOMT2 mediates 3,5,4′-tri-methylation as well as 3,4′-di-methylation of stilbene (resveratrol) , which we used for constructing pterostilbene-producing E. coli (BL21/PTS) in this study, and that from S. bicolor (SbOMT3) have 3,5-di-methylation activity toward resveratrol [Catharanthus roseus (CrOMT2) can catalyze di-methylation of flavonol and flavanol (myricetin and dihydromyricetin) at the positions 3′ and 5′ [Mesembryanthemum crystallinum (PFOMT) has also been reported [Triticum aestivum (TaOMT2) exhibits 3′,5′-di- and 3′,4′,5′-tri-methylation activities toward 5,7,3′,4′,5′-pentahydroxyflavone (tricetin) [OMTs that can catalyze the di-methylation reaction of stilbene and flavonoids have rarely been reported. For example, the OMT from veratrol , 22. Altveratrol , the OMT′ and 5′ . The di-reported . To our ricetin) . Thus, tE. coli–Streptomyces cocultivation system for carrying out microbial production of di- and tri-methylated flavonoids for the first time. The cocultivation of BL21/NRG and DHS2001/SaOMT2 produced 5-hydroxy-7,4′-dimethoxyflavanone, 4′-hydroxy-5,7-dimethoxyflavanone, and 5,7,4′-trimethoxyflavanone at titers of approximately 15 mg/L, 34 mg/L, and 11 mg/L, respectively  and S. venezuelae (40 h) was set in consideration of the different growth rates of these microbes before optimization of the mixing ratio. Therefore, further experimental optimization of the coculture conditions such as optimal substrate concentration and preculture period would improve the current titers. Metabolic engineering strategy such as the coexpression of SAM (a methyl donor) synthetase in the OMT-expressing S. venezuelae [E. coli [E. coli and SaOMT2-expressing S. venezuelae developed in this study proves to be an efficient biological tool for facilitating high-yield production of multi-methylated phenylpropanoid compounds.Further optimization of culture conditions and metabolic engineering of both bacteria . Additionezuelae  as well [E. coli  would alS. avermitilis (SaOMT2) capable of multi-methylation of flavanone, flavone, and stilbene was discovered when SaOMT2 was expressed in S. venezuelae. The development of a cocultivation system based on phenylpropanoid-producing E. coli and SaOMT2-expressing S. venezuelae by exploiting this diverse regiospecificity of SaOMT2 resulted in efficient microbial production of di- and tri-methylated phenylpropanoids including 5-hydroxy-7,4′-dimethoxyflavanone, 4′-hydroxy-5,7-dimethoxyflavanone, 5,7,4′-trimethoxyflavanone, 5-hydroxy-7,4′-dimethoxyflavone, 5,7,4′-trimethoxyflavone, and 3,5,4′-trimethoxystilbene for the first time. Further optimization and application of this cocultivation strategy toward various phenylpropanoid compounds will facilitate the development of the abovementioned valuable compounds as pharmaceuticals and nutraceuticals.The previously unknown broad regiospecificity of OMT from Escherichia coli DH5α was used for routine subcloning, whereas E. coli BL21(DE3)  was used as a heterologous host for expressing the recombinant SaOMT2 and producing pterostilbene, naringenin, and apigenin. pGEM T-easy  vector was used for subcloning. The gene expression vectors for E. coli strain, namely pCDFduet-1 and pET32a, were purchased from Novagen . The pCDFduet-amp plasmid was prepared by inserting an ampicillin resistant marker for E. coli–Streptomyces coculture. E. coli strains were grown in LB medium [S. avermitilis ATCC 31267 was obtained from the American Type Culture Collection . S. venezuelae DHS2001, in which the native pikromycin polyketide synthase gene was deleted [Streptomyces heterologous host. S. venezuelae mutants were grown in R2YE liquid medium [E. coli–Streptomyces shuttle vector pSE34 containing a strong constitutive ermE* promoter plus a thiostrepton resistance marker [Streptomyces heterologous host. For the expression of phenylpropanoid biosynthetic genes in an E. coli heterologous host, the Os4CL, VvSTS, PeCHS, MtCHI, PcFNS, VvROMT, and codon-optimized SaOMT2 genes were synthesized from Genotech . All bacterial strains and plasmids used in this study are listed in Table  deleted , was used medium  at 30 °CS. avermitilis was isolated using a PureHelix™ Genomic DNA prep kit from NanoHelix . PCR was performed using prime STAR® GXL DNA polymerase from Takara . Ampicillin, thiostrepton, IPTG, SAM, 4-coumaric acid, resveratrol, pterostilbene, 3,5,4′-trimethoxystilbene, naringenin, sakuranetin, apigenin, genkwanin, 5,7,4′-trimethoxyflavone, and formic acid were obtained from Sigma-Aldrich . Desoxyrhapontigenin , 5-hydroxy-3,4′-dimethoxystilbene, 5-hydroxy-7,4′-dimethoxyflavanone, 4′-hydroxy-5,7-dimethoxyflavanone, and 5,7,4′-trimethoxyflavanone were purchased from Carbosynth . HPLC-grade acetonitrile (MeCN), ethyl acetate (EA), methanol (MeOH), and water were acquired from JT Baker . All other chemicals were of the highest available purity grade.Restriction endonucleases and T4 DNA ligase were purchased from New England BioLabs . The genomic DNA from S. venezuelae, a pSE-SaOMT2 plasmid was constructed. The SaOMT2 gene was amplified from the S. avermitilis genomic DNA using PCR and by employing primers SaOMT2-1_F/SaOMT2-1_R , yielding the corresponding recombinant strain BL21/SaOMT2-H , resulting in the formation of BL21/PTS , resulting in the formation of BL21/NRG and BL21/NRG-SaOMT2 , thereby resulting in the formation of BL21/APG and BL21/APG-SaOMT2 , histidine-tagged recombinant proteins were eluted using NPI-500 buffer . The purified proteins were analyzed by 12.5% SDS-PAGE and visualized using Coomassie brilliant blue. Typically, approximately 11 mg/L and 9 mg/L of purified SaOMT2 proteins were obtained from DHS2001/SaOMT2-H and BL21/SaOMT2-H, respectively.The harvested cells were resuspended in Tris–HCl (pH 7.0) buffer solution and lysed by sonication using a VCX 500 from Sonics & Materials, Inc. . The cell lysates were separated by centrifugation for 30 min at 4 °C, and then the supernatant was purified using Ni2, and 100 μM SAM; this mixture was allowed to react for 12 h at 37 °C. The reaction was stopped by adding two volumes of EA and centrifuged at 13,000 rpm for 10 min. After the organic phase was evaporated to dryness, the residue was dissolved in MeOH. A portion of this solution was analyzed by UPLC–qTOF–HR-MS.The enzyme reaction involving purified SaOMT2 was carried out using a mixture comprising 25 mM Tris–HCl (pH 7.0) supplemented with 1.23 mM of purified SaOMT2, 100 mM apigenin, 100 μM MgCl600 value of 0.2 and cultivated until an OD600 of 0.7–0.8 was reached. At that point, the IPTG was added into the culture broth until the final concentration became 1 mM and continuously incubated at 30 °C for another 3 h. The cells were harvested at 4 °C by centrifugation (10 min at 6000 rpm) and resuspended with 15 mL of fresh R2YE medium. The culture was supplemented with 1.2 mM 4-coumaric acid plus 1 mM IPTG and further incubated for 18 h at 30 °C.The seed culture broth that was subjected to overnight incubation was subsequently inoculated into 30 mL LB medium at an ODE. coli–Streptomyces cocultivation, the seed cultures of E. coli mutants , which were subjected to overnight incubation, were inoculated into 100 mL LB medium at an OD600 value of 0.2. The culture broth was incubated at 37 °C until an OD600 of 0.7–0.8 was reached. Subsequently, the biosynthetic genes were induced with 1 mM IPTG, and the cells were grown at 30 °C for another 3 h. Meanwhile, 1 mL of 36 h-old cultivated DHS2001/SaOMT2 was inoculated into 100 mL of R2YE medium and grown at 30 °C for 48 h. E. coli and Streptomyces cells were mixed volumetrically according to the indicated inoculation ratios. The cocultures were fed with 1.2 mM of 4-coumaric acid plus 1 mM IPTG and allowed to grow for different periods at 30 °C.For methylated phenylpropanoid production using E. coli and S. venezuelae mutants or their cocultures were extracted and partitioned twice using an equivalent volume of EA in a flask, and the EA layers of each culture were combined and concentrated in vacuo. Each EA extract was dissolved in MeOH and then analyzed by UPLC–qTOF–HR-MS.The cultures of ® G2S Q-ToF mass spectrometer coupled with a Waters Acquity UPLC® system equipped with an Xbridge® C18 column  consisting of an Acquity I-Class system. Gradient elution using solvent A (0.1% TFA) and solvent B (80% aq. MeCN with 0.1% TFA) as the mobile phase at a flow rate of 0.2 mL/min at 40 °C was performed. The MS system was operated in ESI with a positive ionization mode. The typical operating parameters were as follows: analyzer, resolution mode; capillary voltage (volt.), 3.0 kV; sampling cone volt., 30 V; source temperature, 120 °C; source offset, 80; desolvation temperature, 400 °C; cone gas flow, 10 L/h; desolvation gas flow, helium collision gas (600 L/h). The analyzer was operated with an extended dynamic range at 60,000 resolution (FWHM at m/z 556) and with an acquisition time of 0.1 s. Leucine enkephalin  as a lockspray was infused at a rate of 5 μL/min for mass correction. Mass spectra were acquired over a scan range of 50–600 amu with a scan time 0.1 s.UPLC–qTOF–HR-MS analysis of the flavonoid and stilbene analogs was performed on a Waters XEVOAdditional file 1. Figures S1–S5 and Table S1.


Ralstonia solanacearum. Previous studies found that silicon (Si) can increase tomato resistance against R. solanacearum, but the exact molecular mechanism remains unclear. RNA sequencing (RNA-Seq) technology was used to investigate the dynamic changes of root transcriptome profiles between Si-treated (+Si) and untreated (−Si) tomato plants at 1, 3, and 7 days post-inoculation with R. solanacearum. The contents of salicylic acid (SA), ethylene (ET), and jasmonic acid (JA) and the activity of defense-related enzymes in roots of tomato in different treatments were also determined. The burst of ET production in roots was delayed, and SA and JA contents were altered in Si treatment. The transcriptional response to R. solanacearum infection of the +Si plants was quicker than that of the untreated plants. The expression levels of differentially-expressed genes involved in pathogen-associated molecular pattern-triggered immunity (PTI), oxidation resistance, and water-deficit stress tolerance were upregulated in the Si-treated plants. Multiple hormone-related genes were differentially expressed in the Si-treated plants. Si-mediated resistance involves mechanisms other than SA- and JA/ET-mediated stress responses. We propose that Si-mediated tomato resistance to R. solanacearum is associated with activated PTI-related responses and enhanced disease resistance and tolerance via several signaling pathways. Such pathways are mediated by multiple hormones , leading to diminished adverse effects  normally caused by R. solanacearum infection. This finding will provide an important basis to further characterize the role of Si in enhancing plant resistance against biotic stress.Bacterial wilt is a devastating disease of tomato caused by soilborne pathogenic bacterium  Ralstonia solanacearum is a destructive disease that limits tomato (Solanum lycopersicum) production throughout the world . . R. solaTotal RNA from Si-treated root samples  and no−Si treated samples  was extracted separately using Column Plant RNAOUT  in accordance with the manufacturer’s instructions. For each sample, equal amounts of RNA (20 μg) extracted from the three plants were pooled for cDNA library construction and qRT-PCR analysis. The construction of cDNA libraries and RNA-Seq was performed by Genedenovo Bio-Tech Co., Ltd. . The library was sequenced on an Illumina HiSeq™ 4000 platform with paired-end sequencing reads (2 × 100 bp). 2-fold change >1 and the false discovery rate < 0.05. The expression patterns of DEGs were analyzed using Short Time-series Expression Miner (STEM) [p-value <0.05 were considered significantly expressed. GO terms and KEGG pathways with corrected p-value ≤0.05 were considered significantly enriched. Pearson correlation coefficient analysis and PCA on all samples were performed by R package (Version 2.15.3).Raw sequences with adaptors and unknown nucleotides above 5% or those that were of low quality were removed to obtain clean reads. The clean reads were mapped to the tomato reference genome  using Tor (STEM) . The clu−1; final column temperature, 250 °C for 5 min; carrier gas (nitrogen) flow rate, 40 mL min−1; hydrogen flow rate, 40 mL·min−1; air flow rate, 400 mL min−1; temperature of injection and detection ports, 250 °C. The ET, SA, and JA contents in fresh root tissues of each treatment at 1, 2, 3, and 7 dpi were measured. For determination of ET, 0.5 g of roots were transferred to 2 mL glass vials, and all bottles were sealed for 8 h. Then, 1 mL of gas was obtained by an air-tight syringe to analyze ET content by an Agilent 6890N gas chromatograph system with a flame ionization detector using an HP5 column (30 m × 0.25 mm). The operating conditions of the GC were as follows: injection volume, 1 mL; initial column temperature, 50 °C for 3 min; temperature increase rate, 15 °C·min2O: 1% acetic acid), and then stored at 4 °C for 24 h. The supernatants were filtered using nylon syringe filters (0.22 μm), and a Waters ACQUITY UPLC system  was used with a Waters ACQUITY UPLC BEH C18 column . The mobile phases consisting of Mobile Phase A (water with 1% formic acid) and Mobile Phase B (methanol with 1% formic acid) were used with a gradient elution of A/B (v/v) from 60:40 , 60:40–40:60 (0.5–4 min), 40:60–60:40 (4 to 4.5 min), and 60:40  at a flow rate of 0.40 mL/min. The column temperature was 35 °C, and the injection volume was 10 μL. The UPLC system was coupled to Waters Quattro Premier XE in the multiple reaction monitoring mode. The electrospray ionization (ESI) source was operated in both positive and negative ion modes. During the first time segment (0–4 min), SA was analyzed in the positive ESI mode. Within the second time segment (4.2–5.5 min), JA was analyzed in the negative ESI mode. The operational parameters of ESI source were as follows: capillary potential = 3000 V, source temperature = 150 °C, desolvation temperature = 450 °C, cone gas flow = 50 L·h−1, desolvation gas flow = 600 L·h−1, and drying gas flow = 15 L·min−1. The precursor ions, product ions, and MS/MS parameters are displayed in SA and JA contents were determined via ultra-performance liquid chromatography-tandem mass spectrometry. For extraction of SA and JA, root tissues (0.5 g) were ground, suspended in 1 mL of extraction solvent , 4.8 μL of sterile water, and 10 μL of qPCR master mix in the ABI Step One Plus Real-Time PCR System . The amplification cycling program was as follows: 90 s at 95 °C, followed by 40 cycles of 95 °C for 5 s, 60 °C for 15 s, and 72 °C for 20 s. All primers for qRT-PCR are listed in -△△Ct method [qRT-PCR was performed to validate the RNA-Seq results for 10 gene transcripts. The purified RNA (1 μg) was reverse transcribed to cDNA using the FastQuant RT kit (with gDNase) . All qRT-PCR reactions were performed in a 20-μL volume composed of 4 μL of cDNA, 0.6 μL of each primer (10 µM µLt method . qRT-PCR


Glucose-6-Phosphate dehydrogenase (G6PD) deficiency is the most common enzymopathy worldwide, no reliable bedside diagnostic tests to quantify G6PD activity exist. This study evaluated two novel quantitative G6PD diagnostics.Participants with known G6PD activity were enrolled in Bangladesh. G6PD activity was measured by spectrophotometry, Biosensor  and STANDARD G6PD . G6PD activity was measured repeatedly in a subset of samples stored at room temperature and 4°C.158 participants were enrolled, 152 samples tested by BS, 108 samples by SG and 102 samples were tested by all three methods. In comparison to spectrophotometry BS had sensitivity and specificity of 72% (95%CI: 53–86) and 100% (95%CI: 97–100) at 30% cut off respectively, while SG had a sensitivity of 100% (95%CI: 88–100) and specificity of 97% (95%CI: 91–99) at the same cut off. The sensitivity and specificity at 70% cut off activity were 71% (95%CI: 59–82) and 98%  respectively for BS and 89% (95%CI: 77–96) and 93% (95%CI: 83–98) respectively for SG. When an optimal cut-off was applied the sensitivity of the BS at 70 cut off rose to 91% [95%CI: 80–96] and specificity to 82% [95%CI: 83–89]; a diagnostic accuracy comparable to that of the SG (p = 0.879). G6PD activity dropped significantly  within 24 hours in samples stored at room temperature, but did not fall below 90% of baseline activity until day 13 , p<0.001).BS and SG are the first quantitative diagnostics to measure G6PD activity reliably at the bedside and represent suitable alternatives to spectrophotometry in resource poor settings. If samples are stored at 4°C, G6PD activity can be measured reliably for at least 7 days after sample collection. The latter maintains sufficiently high concentrations of glutathione, essential for binding radicals and protecting human cells from the adverse effects of oxidative stress  and specificity of 86% [95%CI: 73–94]; At the reference laboratory, the Hb concentration was measured by CBC in 158 samples with a mean concentration of 12.9 g/dL (95%CI 12.6–13.1).s = 0.861, p<0.001), with a mean difference of -0.48 g/dL (range: -3.50 to 2.60), corresponding to a variation of 4% of the mean Hb value measured by MHD-1  with a mean difference of 0.4 g/dL (range: -2.8 to 1.6), corresponding to a variation of 3%;  at baseline to 7.6 U/gHb (95%CI: 6.7–8.5) at 24 hours; a mean fractional fall of 5.4% (p = 0.022). When stored at 4°C the overall G6PD activity fell from a mean of 7.9 U/gHb (95%CI: 7.0 to 8.8) to 7.5 U/gHb (95%CI: 6.6 to 8.4) within 3 days (p = 0.001), a fractional fall of 6%. A fall of greater than 10% was not observed at room temperature within the first 24 hours, but at 4°C was observed after 13 days . In a miWe present data from two novel devices to quantify G6PD activity at the point of care. Each device was compared with spectrophotometry using kits from Pointe Scientific and both devices showed high correlation  with theThe performance of the SG test was assessed under both field and reference laboratory conditions. In the laboratory SG showed an almost perfect correlation with spectrophotometry at 30% cut-off activity, which represents the threshold currently applied to guide hypnozoitocidal primaquine treatment . Areas uThe SG was also evaluated under field conditions and showed a consistent performance as to that in the laboratory. No significant differences in sensitivity and specificity at 70% and 80% cut-off activity between field and laboratory results were found. Furthermore, there was excellent repeatability of field SG results. Indeed, a single measurement in the field reliably identified 9 out of 10 individuals with an enzyme activity less than 70%.The BS determines G6PD activity by measuring the electrochemical properties of a blood sample whereas the SG and spectrophotometry determine G6PD activity through a colorimetric reaction. The BS device was simple to use, with the sample collected directly from a fingerpick and dragged into the measuring chamber by capillary forces. In contrast the SG requires a specified amount of blood to be pipetted to a buffer and the blood buffer solution subsequently needs to be transferred with a pipette to the test field of the machine. The BS device costs approximately 670 USD, with each strip costing 3.40 USD, while the SG device costs approximately 380 USD with single use strips costing 3 USD per test. While the BS requires a separate Hb measurement and results must be normalized manually, the SG has an integrated Hb measurement, providing normalized G6PD activity and a Hb measurement from the same sample and strip.Both methods for measuring Hb performed well against the reference method (CBC), with a mean difference of less than 0.5 g/dL, an acceptable degree of accuracy for most clinical applications and thus suitable alternatives to other PoC Hb measurement devices. Comparable findings for the MHD-1 had been reported earlier, underlining its consistent performance .Our study addressed the practical application of G6PD testing and the duration for which samples could be stored before testing. At room temperature G6PD activity derived from spectrophotometry remained stable initially but had decreased by 5% at 24 hours. The drop in G6PD activity appeared to be independent of the underlying G6PD activity, suggesting that the absolute decay is not G6PD variant specific. At 4°C, G6PD activity did not fall by more than 10% until after 9 days of storage. Hence the current recommendation that samples should be stored at 4°C and processed within 7 days after sample collection is conservative but valid.P. vivax malaria.In conclusion, significant progress has been made to develop point of care quantitative measurement of G6PD activity. Our study presents field data gathered for two novel diagnostics, both of which performed well under field and laboratory conditions with comparable accuracy. These tests offer, for the first time, reliable alternatives to spectrophotometry in settings with poor laboratory infrastructure, and have potential to facilitate the safe roll out of the radical cure of S1 Figr = 0.0030; p = 0.970, n = 158.(PDF)Click here for additional data file.S2 FigRepeatability of two measurements of the STANDARD G6PD TEST in the field- a) Scatter plot and b) Bland–Altman plot.a) rs = 0.9025; p<0.001, n = 106 b) Mean difference: - 0.66 U/gHb, 95% LoA: -2.04 to 3.36 U/gHb (grey shaded area).(PDF)Click here for additional data file.S3 FigComparison of the STANDARD G6PD Test (SG) in the lab and field a) Scatter plot and b) Bland-Altman plot.a) rs = 0.8765; p<0.001, n = 106 b) Mean difference: -0.33 U/gHb, 95% LoA: -3.29 to 2.63 U/gHb (grey shaded area).(PDF)Click here for additional data file.S4 FigComparison of the STANDARD G6PD Test (SG) in the field against spectrophotometry a) Scatter plot and b) Bland-Altman plot.a) rs = 0.9122; p<0.001, n = 106 b) Mean difference: -1.18 U/gHb, 95% LoA: -4.20 to 1.84 U/gHb (grey shaded area).(PDF)Click here for additional data file.S5 FigComparison of the MHD-1 in the lab against CBC-HB a) Scatter plot and b) Bland-Altman plot.a) rs = 0.8614; p<0.001, n = 158 b) Mean difference: 0.48 g/dL, 95% LoA: -1.98 to 1.01 g/dL (grey shaded area).(PDF)Click here for additional data file.S6 FigComparison of the SG-Hb in the lab against CBC-HB a) Scatter plot and b) Bland-Altman plot.a) rs = 0.8892; p<0.001, n = 108 b) Mean difference: 0.37 g/dL, 95% LoA: -0.80 to 1.54 g/dL (grey shaded area).(PDF)Click here for additional data file.S7 FigGrey shaded area indicates 10% of measurement, equivalent to max. variation of spectrophotometry.(PDF)Click here for additional data file.S1 Data(XLSX)Click here for additional data file.


Leishmania spp. In this work, a sesquiterpene lactone-rich preparation—a leaf rinse extract (LRE) from Tithonia diversifolia—was tested against promastigote forms of L. braziliensis. The results revealed that the LRE is a rich source of potent leishmanicidal compounds, with an LD50 value 1.5 ± 0.50 µg·mL−1. Therefore, eight sesquiterpene lactones from the LRE were initially investigated against promastigote forms of L.braziliensis. One of them did not present any significant leishmanicidal effect (LD50 > 50 µg·mL−1). Another had a cytotoxic effect against macrophages (4.5 µg·mL−1). The five leishmanicidal compounds with the highest level of selectivity were further evaluated against intracellular parasites (amastigotes) using peritoneal macrophages. Tirotundin 3-O-methyl ether, tagitinin F, and a guaianolide reduced the internalization of parasites after 48 h, in comparison with the negative control. This is the first report on sesquiterpene lactones that have potent leishmanicidal effects on both developmental stages of L. braziliensis.Natural compounds represent a rich and promising source of novel, biologically active chemical entities for treating leishmaniasis. Sesquiterpene lactones are a recognized class of terpenoids with a wide spectrum of biological activities, including activity against  Leishmania. Leishmania parasites have a dimorphic life cycle altering between an extracellular promastigote and an intracellular amastigote form [Leishmaniasis is a zoonosis caused by an intracellular parasite belonging to the genus ote form . This diote form ,2. In thote form ,4. The sote form . Among tote form ,7,8,9.Tithonia diversifolia (Hemsl.) A. Gray (Asteraceae), popularly known as Mexican sunflower or “margaridão”, has been used in folk medicine as an anti-inflammatory, and for treating diabetes, microbiological infections, snakebites, and malaria, among other ailments [Leishmania species  [L. braziliensis.ailments ,11,12. Tailments ,11,13, wailments ,15,16, iailments ,17. Diffonensis) ,18, and in silico studies [T. diversifolia dichloromethane leaf rinse extract and the fact that its STL act against both the promastigote and amastigote forms of L. braziliensis.Based on the aforementioned lack of research, and as part of our ongoing efforts to discover novel natural compounds with antiparasitic properties ,9,19,20, studies , we repoT. diversifolia dichloromethane leaf rinse extract (LRE) presents strong in vitro antileishmanial activity. Notably, LRE is a rich source of STL. During the rinse process (with organic solvents), these compounds are extracted from glandular trichomes located on the leaf surface; the trichomes are where STL are biosynthesized and stored [50 value of 1.5 ± 0.50 µg·mL−1. Through scanning electron microscopy (SEM), morphological analysis of promastigotes revealed noticeable differences between the treated parasites and the control group (−1 (6 h incubation). When parasites were incubated with LRE under these conditions, they lost two major promastigote characteristics: (i) their fusiform morphology changed to a rounded shape and; (ii) the flagellum in the majority of the cells was missing. All this information suggests that the LRE is a potential source of natural compounds with leishmanicidal activity. We accordingly decided to investigate the antileishmanial potential of the LRE’s main metabolites, i.e., STL.We show, for the first time, that d stored ,13. The ol group ; treatedL.braziliensis (Table). STL 3 was the only compound that did not show an in vitro leishmanicidal effect in the evaluated concentrations, and displayed an LD50 value that was higher than 50 µg·mL−1 , the major STL present in LRE [1 , did not show significant toxicity in macrophages, and displayed a high level of selectivity  that are found in the sandfly’s bite site and are phagocytosed. Inside of parasitophorous vacuoles, metacyclic promastigotes transform into aflagellate amastigotes. The replication of these forms promotes the rupture of host cells, thus releasing infective amastigotes. This promotes the massive infection of macrophages, so as to continue the transmission cycle ,22.2 and 5–8) were evaluated at their LD50 concentrations. The results showed that, when compared to negative control , STL 5, 6, and 7 were able to reduce significantly (p < 0.05) the infection index after 48 h of treatment .STL T. diversifolia leaves were collected in May, 2002, by S.R.A. in Ribeirão Preto, SP, Brazil. Plant material was identified by Prof. J. N. Nakajima . A voucher specimen (FBC #126) was deposited at the SPFR herbarium . Air-dried whole leaves (3.7 kg) were rapidly rinsed with CH2Cl2 to yield 82 g of light-yellow extract (LRE). Detailed information about the fractionation of the LRE via different chromatographic techniques and the purification of STL has been previously reported [1, 88.0 mg), 1β,2α-epoxytagitinin C , 3-hydroxy-8β-isobutyryloxydehydroleucodin , tirotundin , tirotundin 3-O-methyl ether , tagitinin F , 4β,10α-dihydroxy-3-oxo-8β-isobutyryloxyguai-11(13)-en-6α,12-olide , and tagitinin A .reported . The LREL. braziliensis (H3227 MHOM/BR/94/H-3227) promastigotes were originally isolated from a human case of mucocutaneous leishmaniasis in the state of Ceará, Brazil. The cultures were maintained at 25 °C in M199 medium supplemented with 10% FBS , 2% human urine, 100 mM adenine, 10 mg·mL−1 hemin, 40 mM HEPES (pH 7.4), 50 units·mL−1 penicillin, and 50 mg·mL−1 streptomycin.Cultures 2HPO4, 2.7 mM KCl, 1.5 mM KH2PO4, pH 7.0). The peritoneal exudate cells were centrifuged at 400 ×g for 10 min and the pellet was incubated at 37 °C for 15 min in Ammonium-Chloride-Potassium (ACK) lysis solution  for removal of the remaining red blood cells. Macrophages were pelleted at 400 ×g for 10 min, washed with PBS, and re-suspended in RPMI 1640 medium  supplemented with 10% heat-inactivated FBS and 1% streptomycin/penicillin at 1 × 106 cells·mL−1.Thioglycolate-elicited peritoneal macrophages were obtained from BALB/c mice (weighing 20–25 g) by injection of 1 mL of 3% thioglycolate three days prior to peritoneal lavage with 10 mL of cold PBS  and the plate was incubated at 26 °C for 72 h. After that, 100 μg of 3--2,5-diphenyltetrazolium bromide (MTT), dissolved in 10 μL of sterile PBS, were added per well and the plates were incubated at 37 °C for 4 h. The plates were centrifuged at 4,000× g for 5 min and the supernatant was removed. The precipitated formazan was dissolved with 100 µL of dimethyl sulfoxide (DMSO) and absorbance was measured at 492 nm. The assays were evaluated in triplicate and repeated at least twice.The antileishmanial assay was carried out according to the procedures outlined by Dutta et al. . The LRE6 macrophages and 5 × 107 parasites (10:1 parasites per cell ratio) in 10 mL RPMI 1640 medium were incubated at 27 °C in a polypropylene tube for 3 h. Non-internalized parasites were removed by three centrifugation cycles at 50 ×g. The infected macrophages were plated on glass coverslips (13 mm diameter) in 24-well plates and incubated for 1 h at 34 °C; non-adherent macrophages were removed by washing (three times) with PBS. Infected macrophages were treated with STL 2 and 5–8 diluted in RPMI medium in the concentration previously determined as the LD50 for promastigotes and then incubated for 48 h at 34 °C in 5% CO2. Untreated infected macrophages were used as control. The coverslips were washed with PBS, stained with Panoptic stain , dried and mounted on glass slides with Tissue-Tek mounting medium . Three hundred macrophage cells per experiment were inspected through bright-field microscopy. The data was expressed as infection index (percentage of infected macrophages by the average number of amastigotes per macrophage) [The antiamastigote assay was carried out as previously described ,25. Brie5 cells/well) were seeded in a 96-well microtiter plate and incubated at 37 °C in a 5% CO2 atmosphere for 18 h. After that, the media were removed and adhered cells were exposed to the compounds (exposure concentrations ranged from 0.12 to 50.0 µg·mL−1) for 48 h under the same incubation conditions. The control wells without STL (untreated cells) were used as controls and were considered to be 100% viable cells. Cellular viability was determined using the MTT assay . Each assay was performed in triplicate with two independent experiments.Macrophages (2 × 1050) values were calculated using dose–response curves in OriginLab software .Data represent the mean number (±SD) of triplicate samples from two independent assays. The 50% lethal dose (LD8) forms of L. braziliensis were grown in the presence or absence of LRE from T. diversifolia (10 μg·mL−1 for 6 h). Cells were attached on 13 mm round coverslips previously coated with Biobond  following the manufacture’s instructions. Cells were fixed in 2.0% glutaraldehyde  for 2 h at RT. Next, they were submitted to a post-fixation process with 1% OsO4 (Electron Microscopy Sciences) for 2 h, rinsed in milli-Q water, and incubated with saturated thiocarbohydrazide (Electron Microscopy Sciences) for 10 min, followed again by 1% OsO4. This step was repeated twice. The promastigotes were dehydrated with a graded series of ethanol and were critically point-dried with liquid CO2 in a BAL-TEC-CPD 030 Critical-Point Dryer . Subsequently, the coverslips were mounted on aluminum stubs with silver paint (Electron Microscopy Sciences) and coated with gold in a BAL-TEC SCD 050 Sputter Coater (BAL-TEC). Finally, promastigotes were examined in a JEOL JSM-5200 scanning electron microscope .Promastigote . These three STL did not show considerable cytotoxicity when tested in peritoneal macrophages. In vivo efficacy studies of these active compounds and a better understanding of their mechanisms of action in Leishmania should be pursued. The results described herein bring new perspectives in the study of these two STL subtypes and may provide promising leads for the discovery of new agents against leishmaniasis.In this work, among seven leishmanicidal STL