Bronchoscopy was performed 6 h post exposure and lung lavage fluids, i.e., bronchial wash (BW) and bronchoalveolar lavage (BAL), were sequentially collected. Mass spectrometry methods were used to detect a wide array of oxylipins (including eicosanoids), endocannabinoids, N-acylethanolamines, and related lipid metabolites in the collected BW and BAL samples. Six lipids in the human lung lavage samples were altered following biodiesel exhaust exposure, three from BAL samples and three from BW samples. Of these, elevated levels of PGE2, 12,13-DiHOME, and 13-HODE, all of which were found in BAL samples, reached Bonferroni-corrected significance. This is the first study in humans reporting responses of bioactive lipids following biodiesel exhaust exposure and the most pronounced responses were seen in the more peripheral and alveolar lung compartments, reflected by BAL collection. Since the responsiveness and diagnostic value of a subset of the studied lipid metabolites were established in lavage fluids, we conclude that our mass spectrometry profiling method is useful to assess effects of human exposure to vehicle exhaust.The adverse effects of petrodiesel exhaust exposure on the cardiovascular and respiratory systems are well recognized. While biofuels such as rapeseed methyl ester (RME) biodiesel may have ecological advantages, the exhaust generated may cause adverse health effects. In the current study, we investigated the responses of bioactive lipid mediators in human airways after biodiesel exhaust exposure using lipidomic profiling methods. Lipid mediator levels in lung lavage were assessed following 1-h biodiesel exhaust  contains supplementary material, which is available to authorized users. Air pollution contributes substantially to the global burden of respiratory and cardiovascular disease –4. Numer2 (PGE2), PGD2, PGF2α, and 15-hydroxyeicosatetraenoic acid (15-HETE). Oxylipins can also be produced from other PUFAs, a common one being linoleic acid (LA), resulting in compounds like 9-hydroxyoctadecadienoic acid (9-HODE), 13-HODE, 9,10-dihydroxyoctadecenoic acid , and 12,13-DiHOME. These oxylipins are formed via cyclooxygenase (COX), lipoxygenase (LOX), and cytochrome P450 (CYP) pathways  with the ability to influence the activity of the CB1 and CB2 receptors amino]-dodecanoic acid (CUDA) used for recovery calculation purposes, were obtained from Cayman Chemical . Only 9,10,13-trihydroxyoctadecenoic acid (TriHOME) and 9,12,13-TriHOME were obtained from Larodan . Acetonitrile (ACN) and methanol (MeOH) were from Merck . Isopropanol was from VWR PROLABO . Acetic acid was purchased from Aldrich Chemical Company, Inc. . Butylhydroxytoluene (BHT) was from Cayman Chemical  and ethylenediaminetetraacetic acid (EDTA) from Fluka Analytical, Sigma-Aldrich . Glycerol was from Fischer Scientific . All solvents and chemicals were of HPLC grade or higher and ultrapure water was used .2 (range 20–26 kg/m2). Each subject was instructed to have a light, ordinary breakfast, to avoid ham, and to eat as similarly as possible before the two exposures. Subjects were also instructed to refrain from alcohol and caffeine for 24 h pre-exposure, and to abstain from use of anti-inflammatory drugs or dietary supplements during the week preceding the exposure and bronchoscopy.The study design was based on a series of preceding studies, as previously outlined . In shor3 in a human exposure chamber in a controlled randomized, double-blinded, crossover fashion. Biodiesel exhaust emissions were generated from RME in its pure form (B100) by a Volvo engine  using the urban part of the European Transient Cycle, in order to mimic urban driving conditions [2 body surface area, with rest. The bronchoscopy procedure was performed around 6 h after completion of the exposure session, according to a well-established, previously described method [Each subject underwent two exposures, with a minimum 3-week interval Fig. . Each 1-nditions . The cond method . The timd method . A detaiN-acylethanolamines, and other related lipids from BW and BAL samples. In summary, extraction was performed on Waters Oasis HLB cartridges . These were first washed with ethyl acetate (1 mL) and MeOH (2 × 2 mL), then conditioned with 5% MeOH in water (containing 0.1% acetic acid) before loading the sample. Quantitative volumes from each sample (resulting in ∼6 mL BAL and 1–3 mL BW) were spiked with 10 μL internal standard (IS) mixture in methanol containing 50 ng/mL 12,13-DiHOME-d4 and 12(13)-EpOME-d4, 25 ng/mL 9(S)-HODE-d4, PGE2-d4 and TXB2-d4, 800 ng/mL 2-AG-d8, 40 ng/mL PGF2α-EA-d4 and PGE2-EA-d4, 20 ng/mL AEA-d8, OEA-d4, SEA-d3 and PEA-d4, as well as 10 μL antioxidant solution (0.2 mg/mL BHT/EDTA in methanol/water (1:1)). After applying the sample, IS, and antioxidant solution, the SPE cartridge was washed, dried under high vacuum and eluted with 3 mL acetonitrile, 2 mL methanol, and 1 mL ethyl acetate. All three fractions were pooled into polypropylene tubes containing 6 μL of a glycerol solution (30% in methanol) to trap the analytes. Eluates were evaporated using a MiniVac system , reconstituted in 100 μL of MeOH, spiked with 10 μL recovery standard (50 ng/mL CUDA), transferred to vials, and analyzed by LC-MS/MS.A previously reported solid phase extraction (SPE) protocol  was modi18 column , with an injection volume of 10 μL for each run. The eluents in the mobile phase consisted of (A) deionized water (0.1% acetic acid) and (B) acetonitrile/isopropanol (90:10). For oxylipin separation, the following gradient was employed: 0.0–3.5 min 10–35% B, 3.5–5.5 min 35–40% B, 5.5–7.0 min 40–42% B, 7.0–9.0 min 42–50% B, 9.0–15.0 min 50–65% B, 15.0–17.0 min 65–75% B, 17.0–18.5 min 75–85% B, 18.5–19.5 min 85–95% B, 19.5–21.0 min 95–10% B, 21.0–25.0 min 10% B [The Agilent Ultra-Performance LC system (Infinity 1290) coupled to an Agilent 6490 Triple Quadrupole mass spectrometer with an electrospray ionization source (ESI) equipped with the iFunnel Technology  was used. Samples for ionization in positive (endocannabinoid-related lipids) and negative (oxylipins) mode were injected separately. Chromatographic separation was performed on a Waters BEH Cin 10% B . The sepin 10% B . The lasThe ESI-MS/MS conditions were as follows: capillary and nozzle voltage at 4000 and 1500 V, drying gas temperature 230 °C with a gas flow of 15 L/min, sheath gas temperature 400 °C with a gas flow of 11 L/min; the nebulizer gas flow was 35 psi, and iFunnel high and low pressure RF were set at 90 and 60 V (negative mode) and 150 and 60 V (positive mode). Dynamic multiple reaction monitoring (MRM) mode was used with fixed time windows (retention time ±2 min) to profile two transitions per compound , see Tables N-acylethanolamines, and related lipids , and eight for oxylipin quantification -EpOME-d4, 9-HODE-d4, PGE2-d4, PGD2-d4, 5-HETE-d8, 20-HETE-d6 and TXB2-d4). For each native compound, a suitable IS was selected based on structural similarities , m is the slope of the curve, x corresponds to the on-column concentration of the analyte, and b is the y-interception of the calibration curve.The stable isotope dilution method was used to quantify the analytes. Two types of internal standard were used: (i) for quantification purposes, deuterated IS was added before extraction, and (ii) for monitoring the loss of IS, the recovery standard CUDA was added after extraction . The addes Table , ESM. Stes Table , ESM to Basal statistical measures  and receiver-operating characteristic (ROC) curves were performed using the statistical package built into the Graphpad Prism software . For analytes below limit of detection (LOD), i.e., non-detects, half of the LOD value was used in the univariate statistical calculations . BonferrN-acylethanolamines as diagnostic markers for exhaust exposure. ROC curves are non-parametric and consider true negatives (specificity) and true positives (sensitivity) identified at a given cutoff value of the feature under analysis, commonly used in metabolomics studies to avoid misinterpretation of the data [ROC curves were generated to further investigate these findings by evaluating the oxylipins and the data . The are3) or filtered air (sham) exposure. Exposures were performed at two different occasions and in random order with subjects acting as their own controls. Six hours after the end of the exposure, bronchoscopy collection of BW and BAL was performed. Both the exposures and the bronchoscopies were well tolerated by all subjects. Our validated mass spectrometry methods [N-acylethanolamines, and fatty acid glycerol esters in the collected BW and BAL samples . Endocannabinoids and other related lipids (N-acylethanolamines and glycerol fatty acid derivatives) have not previously been reported in human BW or BAL, except in a study by Zoerner et al. [Of the 14 compounds screened in the endocannabinoid metabolome Table , ESM usir et al. , in whicN-acylethanolamine levels due to biodiesel exhaust exposure were assessed using the Wilcoxon matched-pairs signed rank test and the results are shown in Table No compound passed the D’Agostino and Pearson omnibus normality test, even with square root transformation. Shifts in oxylipin and 2α  and 15-HETE , as well as elevated levels of 9-HODE  were associated with biodiesel exhaust exposure , see Fig. 2α  and PGE2 . Among the oxylipins and N-acylethanolamines, there were up to 26 possible comparisons, which corresponded to a threshold significance value of 0.002 (Bonferroni correction for multiple testing). All significant compounds in BAL , but none in BW, reached this level  and 9-HODE , are shown in Fig. 2 , 13-HODE , 12,13-DiHOME , and 9,10-DiHOME . However, 9,10-DiHOME displayed a P value close to the upper limit of significance (α = 0.05) and was not significant according to the Wilcoxon test. In Fig. 2 had a Youden score of >3.22 pM. When analyzing the concentrations of each sample, we found that BAL samples collected after biodiesel exhaust exposure produced PGE2 levels above 3.22 pM in 9 out of 15 BAL samples, while only 1 out of 15 BAL samples contained PGE2 levels above 3.22 pM after filtered air exposure. On the contrary, PGF2α  had a Youden score of <16.2 pM in BW, which suggested that levels below 16.2 pM in BW were indicative of biodiesel exhaust exposure. Indeed, BW samples from biodiesel exhaust exposure contained levels of PGF2α below 16.2 pM in 11 out of 15 samples, while only four BW samples contained PGF2α levels below 16.2 pM after filtered air exposure.We determined the AUC for ROC curves of all metabolites detected in BW and BAL and found that metabolites in BAL that had shifted significantly were far from reaching significance in BW and vice versa. The significant ROCs for metabolites in BW, PGFy-axis) against biodiesel exposure profiles (x-axis) produced similar characteristic variables for BAL (1st quadrant) and for BW (3rd quadrant) for both types of exposure , only oxylipins (CV-ANOVA = 1.1 × 10−6), or only N-acylethanolamines (CV-ANOVA = 0.012), were all significant. Analytes that were responsible for the separation in Fig. N-acylethanolamines responsible for the separation were OEA and SEA which were present at higher concentrations in BW than in BAL . The In this study, we explored the responsiveness of bioactive lipid mediators in human airways to an oxidative challenge comprised of controlled exposure to biodiesel exhaust. Biodiesel use is increasing in importance as a renewable carbon dioxide neutral biofuel, and in the present study RME was employed as being among the most common biodiesel fuel in northern Europe. The findings pinpoint a subset of bioactive lipids with diagnostic value for exposure of exhaust derived from a pure RME-biodiesel fuel (B100), which warrants validation in future studies. Further investigations are needed to study differences in effects from biodiesel and conventional petrodiesel exhaust exposure since the exploratory nature of our study poses a limitation and the results cannot be generalized to other biodiesel blends and origin, nor to petrodiesel.Lung lavage fluids representing proximal vs peripheral lung compartments were collected. The analytes selected for quantification are acknowledged as important for different inflammatory conditions (acute and/or chronic), and a limited number of studies in humans have described a subset of them as being responsive to allergens and air pollution exposure  –45. Our N-acylethanolamines in both BAL and BW: the well-known CB receptor ligand anandamide (or AEA), as well as LEA, OEA, PEA, POEA, and SEA. While only limited information exists on the prevalence of endocannabinoids, N-acylethanolamines, and fatty acid glycerol esters in human lung lavage fluids, studies have shown that eicosanoids and associated oxylipins are present at compartment-dependent concentrations, and that the levels are affected by subway air exposure [Interestingly, we detected six exposure , 44.In the present study, we aimed to investigate air pollution-induced effects at different levels of the airways, as distinct responses have been reported in previous studies following exposure to air pollutants , 46. OneN-acylethanolamines. For instance, the LA epoxides 9(10)-EpOME and 12(13)-EpOME were indicative of more distal lung regions (BAL), while 13-HODE and 15-HETE indicated more proximal lung regions (BW). However, individual oxylipins and N-acylethanolamines did not display the same response to biodiesel exhaust exposure in BW and BAL. The same number of analytes displayed a significant shift in BAL and BW . Of these, PGE2, 12,13-DiHOME, and 13-HODE were significantly elevated in BAL after biodiesel exhaust exposure, using Bonferroni correction for multiple testing. No metabolite from the BW samples reached this level of statistical power. These findings were supported by AUC values from ROC curve calculations of each metabolite, indicating their diagnostic value.Consistent with these previous studies, the current investigation showed compartmental specificity with regard to both oxylipins and 2, a COX metabolite derived from AA, is a well-studied compound [2 is usually considered a pro-inflammatory compound, found at elevated levels in various conditions such as tumor growth and pain [2 is present at higher concentrations in the lung than in plasma and has anti-inflammatory and bronchodilator properties despite being associated with airway irritation and coughing [2 are mediated via activation of the EP4 prostanoid receptor, resulting in suppression of airway irritation, supporting a protective effect PGE2 in the lung [2 has also been shown to dampen the reactivity of dendritic cells [2 induced significant airway microvascular leak in mice and guinea pigs that was mediated via activation of the EP2 and EP4 receptors, implying destructive effects of PGE2 [2 is only one example of the complexity of an adequate inflammatory response that, besides PGE2, also involves other studied eicosanoids and associated bioactive lipid mediators that altogether exhibit coordinated and often opposing actions [2 portrays a protective response in the more distal parts of the lung, where fewer signs of an inflammatory response have been detected than in BW [PGEcompound . PGE2 isand pain , 53. Howcoughing . In a rethe lung . Bronchiic cells . However of PGE2 . The int actions . Our resan in BW , 18, 50.2, the increased levels of 12,13-DiHOME and 13-HODE in BAL also reached the significance threshold, after correction for multiple testing, in response to biodiesel exhaust exposure. Both metabolites derive from LA, but 12,13-DiHOME is produced through the CYP pathway (containing the downstream soluble epoxide hydrolase enzyme), while 13-HODE is produced via the LOX pathway. Contrary to PGE2, these two metabolites have been associated with adverse inflammatory outcomes in the airways. In fact, the elevation of both potentially anti-inflammatory (PGE2) and pro-inflammatory  lipid mediators in BAL in response to biodiesel exhaust exposure illustrates the complicated interplay between inflammatory lipid mediators, as described above. The metabolite 12,13-DiHOME, known as iso-leukotoxin-diol, is produced in leukocytes [Besides PGEukocytes . Studiesukocytes  and elevukocytes . The metukocytes , and speukocytes .2α and 15-HETE concentrations, and increased 9-HODE concentrations in BW in response to biodiesel exhaust exposure. These metabolites derive from AA and LA; PGF2α is a COX metabolite (in part produced from PGE2), while 15-HETE and 9-HODE are LOX metabolites. 15-HETE has antioxidant and pro-resolving properties [Furthermore, the current study suggests decreased PGFoperties  and was operties . Hence, operties , 18, 50.operties . Hence, N-acylethanolamines to investigate the responsiveness to RME biodiesel exhaust exposure. The results showed that individual lipids from different lung compartments displayed a change in levels in response to biodiesel exhaust exposure, and a subset was of diagnostic value. Since lipid mediators are important regulators of a wide variety of physiological responses, and since we have shown the responsiveness and diagnostic value of a subset of them to biodiesel exhaust exposure, they might be useful markers of the impact of conventional fuels as well as new, commercially available biofuels, on human health in future investigations.The effect of biodiesel exhaust exposure on human health has long been overlooked in spite of calls for performing studies in this field. Since novel methods for assessing these health effects are desirable, it is attractive to establish profiling techniques to comprehend the complex signaling networks of bioactive lipids that are imperative in coping with inflammatory events. To that end, we used quantitative mass spectrometry profiling protocols to cover a large array of potential bioactive lipids in human lung lavage fluids. Thereby, we quantified multiple oxylipins (including eicosanoids), endocannabinoids, and ESM 1(PDF 679 kb)ESM 2(XLSX 41 kb)Below is the link to the electronic supplementary material.


Escherichia coli (STEC) may cause haemolytic uraemic syndrome (HUS). Age ≤5 years and presence of stx2a and eae are risk factors for the development of HUS. In this study, we investigated STEC isolates for the presence of adhesins, toxins and molecular risk assessment (MRA) factors to identify virulence genes associated with HUS development. We included non-duplicate isolates from all STEC infections  reported to the Norwegian National Reference Laboratory (NRL) for Enteropathogenic Bacteria from 1992 to 2013. The most common STEC were O157:H7/H− (34%) and O103:H2 (14%). We retrospectively screened the isolates by three multiplex polymerase chain reactions (PCRs) for adhesins (n = 11), toxins (n = 5) and MRA (n = 15). We calculated odds ratios (ORs) and adjusted odds ratios (aORs) for associations with HUS development. On average, isolates were positive for 15 virulence genes (range: 1–24); two toxins (range: 0–4), five adhesins (range: 0–8) and eight MRA genes (range: 0–13). The gene combinations were clustered within serotypes. Isolates from HUS cases were positive for eae and IpfAO26, and negative for saa, eibG, astA, cnf, subA and pic. We identified 11 virulence genes with a significant association to HUS development. Multivariable analyses adjusted for age group and Shiga toxin identified nleH1–2  as an independent risk factor for the development of HUS from an STEC infection. This study demonstrated that the non-LEE effector protein nleH1–2 may be an important predictor for elevated risk of developing HUS from STEC infections. We recommend the NRL for Enteropathogenic Bacteria to consider including nleH1–2 screening as part of routine STEC surveillance.Shiga toxin-producing The online version of this article (doi:10.1007/s10096-017-2974-z) contains supplementary material, which is available to authorized users. Escherichia coli (STEC) is a zoonotic food- and waterborne pathogen of a serious public health concern because of its propensity to cause outbreaks, haemorrhagic colitis and the potentially life-threatening complication haemolytic uraemic syndrome (HUS) , 212 isolates (62%) were positive for stx2  and 91 isolates (43%) were positive for both stx1 and stx2.A total of 218 isolates (64%) were positive for stx1 and/or stx2. The most common toxin genes identified were ehxA (n = 290), StcE/StcEO103 (n = 194) and subA (n = 42). A total of 338 isolates (99%) were identified with adhesion genes, most commonly IpfAO26 (n = 306), eae (n = 252) and ihaA (n = 243). A total of 306 (90%) of the isolates were identified with at least one MRA gene. The most common genes identified were nleB (n = 250), Z2099 (n = 248), nleE and ent/espL2n (n = 247) ; two toxins (range: 0–4), five adhesins (range: 0–8) and eight MRA genes (range: 0–13). A total of 319 isolates (94%) were identified with toxin genes other than eae-positive isolates were negative for aaiC and eibG, in addition to the majority being negative for saa (n = 251), cnf (n = 251), subA (n = 250) and astA (n = 248). All eae-negative isolates were also negative for toxB and nleH1–2, in addition to the majority being negative for cnf (n = 87), nleC (n = 86), nleG2–3 (n = 86) and aaiC (n = 85). All HUS cases were positive for eae.All − isolates were positive for 21 virulence genes (range: 9–24); two toxins (range: 1–4), seven adhesins (range: 4–8) and 11 MRA (range: 0–13). All O157:H7/H− isolates were positive for eae, Ipf1O157/OI-141 and Ipf2O157/OI-154. Conversely, none were positive for saa, aaiC, IpfAO113, ehaA and subA. Major differences between non-sorbitol-fermenting O157 (NSFO157) and SFO157 were seen for ihaA, espP, toxB and ureC, which were absent from all SFO157 (n = 10) and present in almost all NSFO157 . Conversely, cdtB I–IV was present in the majority of SFO157  and in a minority of NSFO157 . Among isolates from patients developing HUS of this serotype (n = 12), all isolates were positive for IpfAO26 and all were negative for eibG, astA, cnf, fyuA and pic. Differences among the NSFO157 and SFO157 developing HUS were seen for ihaA, agn43EDL933, espP and toxB, which were present in all NSFO157 and absent from all SFO157.On average, O157:H7/HIpfAO26, ehaA, efa1/lifA, Z2099, nleE and nleB. Conversely, none of the isolates were positive for aaiC, toxB, IpfAO113, Ipf2O157/OI-154, eibG, cnf, cdtB I–IV, nleH1–2, fyuA and pic. All STEC O103:H2 carried stx1a and none of these isolates were from cases that developed HUS.On average, O103:H2 isolates were positive for 13 virulence genes (range: 11–17); two toxins (range: 1–3), three adhesins (range: 2–7) and eight MRA (range: 6–9). All O103:H2 isolates were positive for eae, iha1, IpfAO113, IpfAO26, ehaA, efa1/lifA, ecf4, Z2099, nleE, nleB, paa, fyuA and ent/espL2n, and all were negative for saa, Ipf1O157/OI-141, Ipf2O157/OI-154, eibG, toxB, astA, cnf, cdtB I–IV, subA, nleC and pic. Among isolates from patients developing HUS of this serotype (n = 4), all isolates were positive for agn43EDL933, ureC, nleH1–2 and nleG2–3, and all were negative for pagC-like.On average, O26:H11 isolates were positive for 19 virulence genes (range: 16–20); one toxin (range: 1–2), seven adhesins (range: 6–7) and 11 MRA (range: 9–12). All O26:H11 isolates were positive for iha1, agn43EDL933, Ipf1O157/OI-141, IpfAO26, ehxA, efa1/lifA, Z2099, nleB, nleE, paa, ureC and ent/espL2n, and all were negative for saa, aaiC, toxB, IpfAO113, ehaA, Ipf2O157/OI-154, eibG, astA, cnf, cdtB I–IV, subA, nleH1–2, fyuA and nleG2–3. One of the O145:H28 isolates was recovered from a case that developed HUS and five O145:H? isolates were recovered from cases that developed HUS. All HUS isolates were positive for stx2a, IpfAO113 and nleH1–2, whereas all non-HUS isolates were negative. Conversely, all isolates from HUS cases were negative for iha1, agn43EDL933, Ipf1O157/OI-141 and StcE/StcEO103, whereas all non-HUS isolates were positive.On average, O145:H28 isolates were positive for 15 virulence genes (range: 14–16); two toxins (range: 1–2), five adhesins (range: 4–6) and eight MRA (range: 7–9). All O145:H28 isolates were positive for −, O103:H2, O26:H11 and O145:H28. Isolates from cases developing HUS were seen distributed between multiple clusters, with certain clusters appearing to be less commonly associated with HUS than others. All isolates from HUS cases (n = 32) were positive for eae and IpfAO26, and none were positive for saa, eibG, astA, cnf, subA and pic. None of the isolates recovered from cases aged between 20 and 60 years (n = 117), were serotyped as O103:H2 (n = 47), positive for stx2b (n = 32), stx2d (n = 3), stx2g (n = 2), stx1c (n = 23) or stx1d (n = 3), or recovered from cases that developed HUS. Univariable analyses identified 11 virulence genes with a significant association with the development of HUS in addition to age ≤5 years and stx2a  and the virulence gene nleH1–2  as independent risk factors for the development of HUS  [eae, ehxA and subtypes of stx1 and stx2, and their association with HUS development.Age ≤5 years) and the presence of an  studies , 20–25.  years an−/H7. The combination of virulence genes differed between NSFO157 and SFO157. We observed, as demonstrated in previous studies, that the mosaic structure of OI-43 in SFO157 isolates lacked the iha1 and ureC genes [espA and toxB were absent from our SFO157 isolates [cdt (8%) compared to SFO157 isolates, which were typically positive (70%). An association of cdt and HUS among eae-negative non-SFO157 has previously been suggested, although the association between cdt and HUS in SFO157 is still unclear [cdt-positive. Also, however, SFO157 isolates not associated with HUS were positive for cdt (75%). In addition to sorbitol fermentation, the presence of stx2a was the marker with the highest positive predictive value (PPV) for O157 association with HUS (29%). A single NSFO157 isolate carried only an stx2c subtype of Stx and was associated with HUS. This isolate displayed a very different virulence gene profile to the other NSFO157 isolates, and was the only isolate negative for nleC, ent/espL2n, pagC-like, nleG2–3, nleH1–2, ureC, paa, nleB, ec4, efa1/lifA and StcE/StcEO103  were positive for ureC and pagC-like [The least number and combination of virulence genes among the most frequent serotypes were observed for O103:H2 isolates. Most O103:H2 isolates were grouped within two major virulence gene clusters, separated only by the presence and absence of or Z2099 , but onlnic STEC , 30, 31.agC-like .efa1/lifA, ent/espL2n, nleB, nleE, pagC-like). A complete OI-122 was seen in 98% of the O157 isolates, with only two isolates negative for efa1/lifA. Among the O103:H2 isolates, OI-122 genes efa1/lifA, ent/espL2n, nle and nleE were seen to be co-located in 98% of the isolates, with pagC-like being only present in two isolates. An absent or truncated pagC may be an indicator for low virulence, as shown earlier [pagC-like is also evident in O26:H11 isolates, all of which were positive for all other OI-122-associated genes. However, contrary to O103 isolates, all O26:H11 isolates that were associated with HUS were negative for pagC-like. In O26:H11 isolates, as seen for O157 isolates, stx2a was the marker with the highest PPV for association with HUS (44%). Isolates of the O145 serogroup with an H? phenotype were more frequently associated with HUS than H28 (71% vs. 5%). Overall, our results indicated that the virulence gene composition varies within each serotype, along with the combination of virulence genes required for an HUS-associated subtype  and the pathogenicity island OI-71 located in nleH1–2 were seen to be significantly associated with the development of HUS. However, in a multivariable logistic regression model adjusted for age and stx2a, only nleH1–2 remained with a significant independent association with HUS (aOR 8.4). nleH1–2 has previously been described as an immune system modulator, functioning through inhibition of the NF-KB activation [Escherichia coli (EPEC) and STEC strains [In our univariable analysis, all of the OI-57 and OI-122 genes together with tivation , 35. Stutivation . Its loc HUS aOR .4. nleH1 strains , 37.nleH1–2 in the detection of STEC isolates recovered from HUS patients was estimated to be 91%, which implied that only three isolates recovered from HUS patients were negative for nleH1–2. Included in these isolates was the stx2c-only NSFO157 isolate from a 66-year-old patient and an O111 isolate recovered from an HUS patient with multi-strain infection where one strain was nleH1–2-positive (carrying stx1a and stx2a) and the other strain was negative (stx1a only). Excluding these two isolates as probable non-HUS-associated, the sensitivity of nleH1–2 increased to 97%, matching the sensitivity of stx2a, although with a lower specificity (56% vs. 80%). The negative predictive value (NPV), when combining age (≤5 years), eae, stx2a and nleH1–2, was estimated to be 97%, indicating that the likelihood of developing HUS was very low if all these factors were negative. The PPV was estimated to be 73%, which was an increase from 68% if we only considered age (≤5 years), eae and stx2a. Also, the specificity increased from 96 to 97% when including nleH1–2, allowing for a more accurate exclusion of non-HUS-associated STEC. The matter of concern was the low sensitivity (75%), which implied that 25% of the STEC isolated from HUS patients in the current study did not harbour this combination of risk factors. However, when excluding the two probable non-HUS-associated isolates, the sensitivity increased to 80%. Overall, the inclusion of nleH1–2 increased our probability of discerning HUS-associated STEC, although other virulence factors and host-specific factors are important when assessing patients at risk of developing HUS.The sensitivity of Our study was limited by the virulence genes selected and the number of isolates tested. A methodological selection bias was present for O157 due to diagnostic challenges and, consequently, underreporting of non-O157 STEC. The low number of isolates and HUS cases prevented a serotype-specific statistical HUS association analysis. Furthermore, we only included STEC isolates in our study, and the prevalence of these virulence genes in other EPEC were not determined and, therefore, a direct association to the development of HUS is likely an overestimation. Also, we did not perform any gene expression analysis to confirm the level of gene translation. Lastly, data on host factors other than age were not available to allow for adjustment in our statistical model.nleH1–2, together with age ≤5 years and stx2a, may contribute significantly in discerning HUS-associated STEC (PPV 73%). The OR of developing HUS from an STEC infection was eight times higher when stains were positive for nleH1–2. Larger studies are required to increase the statistical power of the reported significant associations to enable a better identification of HUS-associated STEC and review infection control guidelines in light of new knowledge. We recommend the Norwegian NRL to include screening for nleH1–2 in routine STEC surveillance to improve the supervision of appropriate infection control measures for sporadic cases and during STEC outbreaks.Our results showed that the non-LEE-encoded immune system modulator Fig. 2n = 340) based on the absence or presence of 33 virulence genes and subtypes of stx1 and stx2. Phylogenetic tree constructed using Dice similarity matrix with a complete linkage cluster analysis method with 100 bootstrap simulations. Isolates of major serotypes are coloured according to the legend. The presence of virulence genes is depicted by a black box under the corresponding virulence gene column. Isolates from cases developing HUS are highlighted with an orange box. (PDF 218 kb)Phylogenetic estimation of the relatedness of STEC isolates


It is regulated by a complex pattern of over 50 different post-translational modifications, including ubiquitination by the E3 ligase MDM2, which leads to its proteasomal degradation. We have previously reported that expression of Bruton’s Tyrosine Kinase (BTK) induces phosphorylation of p53 at the N-terminus, including Serine 15, and increases its protein levels and activity. The mechanisms involved in this process are not completely understood. Here, we show that BTK also increases MDM2 and is necessary for MDM2 upregulation after DNA damage, consistent with what we have shown for other p53 target genes. Moreover, we found that BTK binds to MDM2 on its PH domain and induces its phosphorylation. This suggested a negative regulation of MDM2 functions by BTK, supported by the fact BTK expression rescued the inhibitory effects of MDM2 on p53 transcriptional activity. Indeed, we observed that BTK mediated the loss of the ubiquitination activity of MDM2, a process that was dependent on the phosphorylation functions of BTK. Our data together shows that the kinase activity of BTK plays an important role in disrupting the MDM2-p53 negative feedback loop by acting at different levels, including binding to and inactivation of MDM2. This study provides a potential mechanism to explain how BTK modulates p53 functions. The underlying mechanism is the nuclear export and proteasome-mediated degradation of the polyubiquitinated protein , 20. ThePrevious studies have reported phosphorylation of many residues of p53, including S15, S20, S46, S315, S373, S376, and S392 . PhosphoWe previously found that Bruton’s Tyrosine Kinase (BTK) induces the phosphorylation of p53 at the N-terminus (mainly at serine 15) and that this has an important effect in its protein levels and activity . BTK is Here, we further explore the impact of BTK in the p53 pathway by investigating its effects on MDM2. We found that BTK reinforces its upregulation of p53 activity by phosphorylating and inhibiting MDM2. Our results confirm the relevance of BTK as a novel modulator of p53 activity and provide a mechanistic explanation for its regulatory role in the p53 pathway.We have shown that BTK increases p53 protein levels and activity , which cin vitro kinase assay showed that BTK phosphorylated MDM2 . pCDNA3 was used as empty vector for controls.2. After 24–48 hours, media was changed. Cells were cultured and grown until 70–80% was reached, and transfections were carried with Turbofect , following manufacturer’s instructions. Dilution of plasmids was performed in serum-free medium . Cells were incubated with the plasmid/Turbofect mix for 24–48 hours and then collected. 1.5 μM doxorubicin (Sigma-Aldrich) was added to induce DNA damage. 0.5 μM Ibrutinib  was used to inhibit BTK. 2 μM MG-132 was used to inhibit the proteasome.HCT116 colon cancer cell line (with wt p53), HT1080 osteosarcoma cell line (with wt p53) and human diploid fibroblasts (HDF) were cultured in DMEM and incubated at 37°C with 5% COFor lysate extraction, the medium was removed and plates were washed once with 1x PBS, trypsinized, collected and kept on ice. Cells were ruptured by passing through a syringe 5 times or with sonication, and centrifuged for 15 minutes. The supernatant was transferred into Eppendorf tubes, and protein concentrations were determined using the Bradford protein assay (Fermentas). 10–20 μg of total protein per sample were subjected to 10% or 6% SDS-PAGE and transferred to Immobilon-P membranes (Millipore). Primary antibodies used were: β-actin , p53 , p21 , PUMA , BTK , MDM2  and Streptavidin-Biotinylated HRP Complex .6 cells per sample were used. PCR was performed with 1 μl of DNA. The p53  antibody was used. Primers used: 5′-mdm2 (GGTTGACTCAGCTTTTCCTCTTG), 3′-mdm2 (GGAAAATGCATGGTTTAAATAGCC), 5-Puma: (GCGAGACTGTGGCCTTGTGT), 3′-Puma (CGTTCCAGGGTCCACAAAGT), 5′-p21 (GTGGCTCTGATTGGCTTTCTG), 3′-p21 (CTGAAAACAGGCAGCCCAAG).ChIP assays were performed as previously described . 3 × 106in vivo protein-protein binding assays, cells were transfected with FLAG-MDM2 and BTK, and lysed in lysis buffer  at 4°C. Proteins were immunoprecipitated with anti-IgG , or anti-FLAG antibodies overnight at 4°C, and then protein A/G agarose beads  were added for 4 h with rotation at 4°C. Bound proteins were analysed by Western blotting. For in vitro protein-protein binding assays, cells were transfected with FLAG tagged BTK plasmid (BTK (ΔPH), BTK (ΔK), or BTK (ΔSH3/2)). The cells were lysed and BTK proteins were prepared by immunoprecipitation using anti-FLAG antibody. The purified BTK proteins were mixed with GST-MDM2 individually and incubated for 4 hours with rotation at 4°C. The mixtures were added to glutathione HiCap matrix beads (Qiagen #30900) and incubated for 4 hours at 4°C with rotation. The beads were washed with PBS and eluted, followed by Western blotting using anti-GST and anti-FLAG antibodies.For Cells were transfected with BTK and 6His-Ubiquitin vectors, and after 24 hours treated with MG132 , for 12 hours. Cells were then lysed with 6M Guanidine hydrochloride , and 6His-ubiquitinated proteins were purified on Ni-NTA beads . The bound proteins were eluted and analysed by immunoblotting with p53-specific antibody .100,000 cells per well were seeded in 0.5 ml growth media in 24-well plates. The following day, transfection was performed following the protocol described above. 1.0 μg total plasmid DNA  was used. 24 hours after transfection, media was replaced and part cells treated with 1.0 μM Doxorubicin. 24 hours later, lysates were collected in lysis buffer and stored at –80°C overnight. Prior evaluation, β-galactosidase substrate  and 35 μl β-mercaptoethanol) was prepared and 100 μl was added to 80 μl cell lysate (per well) in a 96-well transparent plate. This was incubated at 37°C for 15 minutes. 20 μl of the cell lysate were transferred to a 96-well opaque white plate and read in a luminometer, using a luciferase kit (BioVision#K801-200), as previously described .RNA purification, cDNA preparation, and qRT-PCR were performed as previously described . PrimersFull length Recombinant BTK , p53, and MDM2 were used in this study, the GST-tagged p53 and MDM2 were obtained by transfecting the appropriate pGEX vectors into BL21 competent and purifying the proteins produced with glutathione HiCap matrix beads (Qiagen #30900), as previously described . 0.5 μg in vitro ubiquitination assay was performed using an ubiquitination kit . The GST-tagged p53 and MDM2 were obtained by transfecting the appropriate pGEX vectors into BL21 competent and purifying the proteins produced with glutathione HiCap matrix beads (Qiagen #30900), as previously described [An escribed  and the Cells were processed and transfected following previously described protocols . Cells wP > 0.05; *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001) was calculated using two-tailed unpaired t tests with Prism 6 (GraphPad) software.All error bars represent the SD. Statistical significance (not significant, ns,


The effects of elevated CO2 concentrations on morphological and anatomical characteristics, and nutrient accumulation have been determined in several plant species. Carrot is an important vegetable and the effects of elevated CO2 on carrots remain unclear. To investigate the effects of elevated CO2 on the growth of carrots, two carrot cultivars (‘Kurodagosun’ and ‘Deep purple’) were treated with ambient CO2  and elevated CO2  concentrations. Under e[CO2] conditions, taproot and shoot fresh weights and the root/shoot ratio of carrot significantly decreased as compared with the control group. Elevated CO2 resulted in obvious changes in anatomy and ascorbic acid accumulation in carrot roots. Moreover, the transcript profiles of 12 genes related to AsA biosynthesis and recycling were altered in response to e[CO2]. The ‘Kurodagosun’ and ‘Deep purple’ carrots differed in sensitivity to e[CO2]. The inhibited carrot taproot and shoot growth treated with e[CO2] could partly lead to changes in xylem development. This study provided novel insights into the effects of e[CO2] on the growth and development of carrots.The CO The eledetected , includi quality . Althouge plants .-1 CO2; -1 CO2; 2 enrichment results in decreased yield. The stomatal conductance has no apparent inhibition under 1000 μmol⋅mol-1 CO2 but gradually decreased in the presence of 3000 μmol⋅mol-1 CO2 for both C3 soybean and C4 maize , a member of the Apiaceae, is an important vegetable crop worldwide because of its edible root and excellent source of vitamins and fibers in the diet. Carrot is a biannual diploid (2n = 2x = 18) with AsA, which is an important material for antioxidant capacity. Compared with the broccoli (3.25–7.64 mg g-1 FW), radish (1.64-3.03 mg g-1 FW) (-1 FW), the AsA levels in ‘Kurodagosun’ is under the range of 0.127–0.163 mg g-1 FW .Carrot ( g-1 FW)  and blueg g-1 FW . BesidesSupplementary Figure S1). ‘Kurodagosun’ is an early maturing variety with an orange root and high resistance against abiotic stress. ‘Deep purple’ is a late maturing variety with a purple and conical root. The seeds of the two carrot cultivars were sown in a chamber at the State Key Laboratory Genetics and Germplasm Enhancement in Nanjing Agricultural University, Jiangsu Province, China . The chamber condition was programmed for a relative humidity of 60–70% with a photoperiod of 16 h light (300 μ mol m-2 s-1) and 8 h dark at day/night temperature of 25/18°C. The plants were grown under normal conditions (387 μmol⋅mol-1 CO2 and 21% oxygen in the atmosphere) for 38 days. Afterward, carrot plants were divided into two groups and transferred to two chambers with similar growth conditions. Simultaneously, the plants were subjected to 400 or 3000 μmol⋅mol-1 CO2, respectively, for another 30 days. To investigate AsA accumulation and expression profiles of AsA-related genes, carrot root samples from the two cultivars were harvested at 10 am at 38, 48, 58, and 68 DAS. Each sample was immediately frozen in liquid nitrogen and stored at -70°C until RNA isolation. Three independent replicates were used for each treatment.‘Kurodagosun’ and ‘Deep purple’ are commonly cultivated carrot cultivars and were selected for analysis in this work . The color of the roots gradually became darker as the roots enlarged. To characterize carrot growth and development, the root/shoot ratio was measured in carrots during different developmental stages . Fresh samples of ‘Kurodagosun’ and ‘Deep purple’ were also harvested for anatomical structure analysis. Samples were cut into about 2 mm-thick slices and then immediately stored in phosphate buffer (pH 7.2) with 2.5% glutaraldehyde solution. Subsequently, the samples were used for safranin O/fast green staining. First, the sheet was dewaxed in xylene for 25 min and dehydrated with different concentrations of ethanol solution. Second, the samples were stained with 1% safranin O for 2 h and dehydrated in a series of ethanol with different concentrations. Afterward, the samples were counterstained with 0.5% green for 15 s, and excess stain was washed out using 95% ethanol and absolute ethanol. The lignin and fiber tissues were stained with red and green, respectively, and observed under a light microscope.The carrots were sampled at 38, 48, 58, and 68 DAS for analysis of the root and shoot biomass accumulation. Carrot roots and shoots were measured from three randomly selected plants per replicate, and the values were derived from the average of three replicate measurements. An orange and purple color first appeared on the taproot surface of ‘Kurodagosun’ and ‘Deep purple’ at 38 DAS, respectively  H3PO4-EtOH, 1 mL of 0.5% (w/v) BP-EtOH, and 0.5 mL of 0.03% (w/v) FeCl3-EtOH were added in order. The mixture was incubated at 30°C for 90 min, and absorbance was measured at 534 nm. To measure the T-AsA levels, 0.5 mL of 60 mM dithiothreitol was added into 0.5 mL of supernatant, and 0.2 M Na2HPO4–1.2 M NaOH was introduced to adjust the pH to 7.0. After reaction for 10 min, the pH was adjusted to 1.5 using 0.3 M TCA. Calibration curves were constructed for both AsA and T-AsA.Determination of AsA levels was performed using the 4, 7-diphenyl-1, 10-phenanthroline method described by Carrot samples from different developmental stages and treatments were ground in liquid nitrogen. The total RNA kit  was used to extract total RNA according to the manufacturer’s instructions. About 10 μg of RNA was used to synthesize first-strand cDNA using the Prime Script RT reagent kit . The cDNA was diluted 18 times for PCR amplification.Arabidopsis and other plant species, a total of 12 genes involved in AsA biosynthesis, degradation, and recycling were identified in CarrotDB (DcPGI), phosphomannose isomerase (DcPMI), GDP-D-manmose pyrophosphorylase (DcGMP), GDP-D-mannose-3′,5′-epimerase (DcGME), GDP-L-galactose phosphorylase (DcGGP), L-galactose-1-P phosphatase (DcGPP), myo-inositol oxygenase (DcMIOX), ascorbate oxidase (DcAO), ascorbic acid peroxidase (DcAPX), monodehydroascorbate reductase (DcMDHAR), dehydroascorbate reductase (DcDHAR), and glutathione reductase (DcGR). A total of six genes  were identified in the L-galactose pathway. DcMIOX were involved in the myo-inositol and D-galacturonic acid pathways, respectively. We have submitted the nucleotide sequences to GenBank, and the corresponding accession numbers were KY347803 (DcGGP), KY347804 (DcGME), KY347805 (DcGMP), KY347806 (DcGPP), KY347807 (DcPMI), KR364573.1 (DcAPX), KY347808 (DcPGI), KY347809 (DcAO), KY347810 (DcDHAR), KY347811 (DcGR), KY347812 (DcMDHAR), KY347813 (DcMIOX) (Supplementary Table S2).Based on the homologous sequences from CarrotDB . These gEx Taq kit, and the primers used for qPCR were designed with Primer 6.0 software (Supplementary Table S1). The data were analyzed by iQ5 software and the iQ5 Real-time PCR System according to the manufacturer’s instruction as follows: 95°C for 30 s, followed by 40 cycles at 95°C for 5 s, 60°C for 30 s, and melting curve analysis. The tubulin gene has been identified as the suitable reference genes for the normalization of gene expression in carrot at different developmental stages . Under the normal condition, the fresh weights of shoots were 15.07 g and 12.27 g at 68 DAS in ‘Kurodagosun’ and ‘Deep purple,’ respectively. Similarly, the fresh weights of roots were 13.40 g and 5.71 g at 68 DAS in ‘Kurodagosun’ and ‘Deep purple,’ respectively. During the carrot development, the fresh weight of shoots and roots were most increased at 58 to 68 DAS. The fresh weights of roots and shoots under a[CO2] treatment were also much heavier than those under elevated CO2 treatment both of two carrot cultivars. In the presence of e[CO2], the shoot biomass showed a 4.31 and 28.3% decrease in ‘Kurodagosun’ and ‘Deep purple’ at 68 DAS, respectively, when compared to plants grown under a[CO2]. The root biomass also decreased by 24.4 and 34.0% in the two cultivars due to e[CO2] at 68 DAS. The root/shoot ratio per cultivar of the a[CO2] and e[CO2] treatments increased slightly over the course of the experiment. Significant changes of the root/shoot ratio within cultivars were found at 58 and 68 DAS .Different morphological characteristics were identified in ‘Kurodagosun’ and ‘Deep purple’ under a, the increased size of VC and lager parenchymal cells were also showed from 48 DAS to 68 DAS in ‘Kurdogosun.’ In addition, the ratio of xylem area to total root area were decreased than that in a[CO2] of ‘Kurdogosun.’ Anatomical structure in ‘Deep purple’ was similar to that in ‘Kurdogosun’ of both a[CO2] and e[CO2] treatments.Safranin-O/fast green staining was used to investigate the anatomical structure of carrot taproot tissues containing lignified cell walls that stain brilliant red. Px, VC, PP, and epidermis were observed at 38 DAS . The AsA contents of both cultivars peaked at 38 DAS and then decreased gradually from 48 to 68 DAS. Compared with the control plants, the levels of AsA and T-AsA under e[CO2] slightly decreased in the two carrot cultivars. ‘Deep purple’ and ‘Kurodagosun’ had the highest AsA levels of 0.163 mg⋅g-1 FW and 0.157 mg⋅g-1 FW, respectively. The highest T-ASA values in ‘Kurodagosun’ and ‘Deep purple’ were about 0.195 mg⋅g-1 FW and 0.193 mg⋅g-1 FW, respectively.Carrot taproots of ‘Kurodagosun’ and ‘Deep purple’ at different developmental stages were used to investigate AsA and T-AsA contents .The results showed that the eight genes involved in the DcPMI peaked at 38 DAS and gradually decreased during root development, which was consistent with AsA accumulation. The expression patterns of DcGMP, DcGGP, DcGPP, DcAO, DcAPX, DcMDHAR, DcDHAR, and DcGR in ‘Deep purple’ were highest at 58 DAS. Among these genes, the transcript levels of DcAO and DcDHAR were similar to those in ‘Kurodagosun’ . Among these nine genes, in the presence of elevated CO2, the different expression levels were showed the five AsA degradation and recycling genes, DcAO, DcAPX, DcMDHAR, DcDHAR, and DcGR, were peaked at 48 DAS. The expression profiles of four genes, namely, DcPGI, DcGMP, and DcGME peaked at 48 DAS and showed the lowest expression levels at 58 DAS. The expression profile of DcMIOX was strongly consistent with the AsA accumulations during carrot development .Under normal conditions, the expression levels of 2] and e[CO2] treatments was provided in Supplementary Figures S1 and S2. The root/shoot ratio of carrot under e[CO2] was lower than that under a[CO2], which was consistent with several studies reported previously , the consistency of increased content of ascorbic acid and enriched CO2 in plants of orange, leaf lettuce, and tomato were demonstrated . Further studies showed that the decreased ascorbate content may be attributed to low levels of photooxidative stress under e[CO2]  is an important enzyme in AsA biosynthetic route, and it is also implicated in sugar metabolism . GME was2], more transcripts of DcAO and DcAPX were detected at 48 DAS when the root suffered from e[CO2] in the two cultivars, whereas the AsA accumulation were decreased. Furthermore, GR also played a key role in other important mechanisms in plants, such as involvement in the cell’s scavenging system for reactive oxygen species by reducing glutathione disulfide (GSSG) to glutathione (GSH).The AsA degradation and recycling pathways also played important roles in the response and adaptation to stress . There a2 . Compared with plants grown under a[CO2], the proportion of xylem region was smaller under e[CO2] in carrot taproot of both cultivars. These results suggested that 3000 μmol⋅mol-1 CO2 inhibited carrot growth and development. The AsA levels decreased in carrots under elevated CO2  treatment. The transcript profiles of most genes  were not well correlated with AsA accumulation under a[CO2] (400 μmol⋅mol-1) and e[CO2] (3000 μmol⋅mol-1) treatments. The results of the present work suggested that the AsA accumulation response to CO2 in carrot taproot resulted from a complex metabolic network, including biosynthesis, recycling, and degradation pathways. The AsA content may also differed in different genotypes of carrot under elevated CO2 treatment.The present study found that the root/shoot ratio in two carrot cultivars decreased in the presence of elevated COA-SX and X-JW conceived and designed the experiments. X-JW, G-LW, FW, Z-SX, Y-ST, and A-SX performed the experiments. X-JW, SS, G-MX, and A-SX analyzed the data. A-SX contributed reagents/materials/analysis tools. X-JW wrote the paper. A-SX, X-LH, and X-JW revised the paper.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


It was characterized by elemental analysis, IR, molar conductivity and thermogravimetric analysis. X-ray crystallography reveals that the Ca(II) coordination polymer belongs to the orthorhombic system, with space group P212121. The geometry of the Ca(II) ion is a distorted CaNO6 pengonal bipyramid, arising from its coordination by four water molecules, one nitrogen atom of 4,4′-bipyridyl molecule, and two oxygen atoms from two L ligands. The complex molecules form a helical chain by self-assembly. The antitumor activity of 1,6-naphthalenedisulfonic acid disodium salt and the Ca(II) coordination polymer against human hepatoma smmc-7721 cell line and human lung adenocarcinoma A549 cell line reveals that the Ca(II) coordination polymer inhibits cell growth of human lung adenocarcinoma A549 cell line with IC50 value of 27 μg/mL, and is more resistive to human lung adenocarcinoma A549 cell line as compared to 1,6-naphthalenedisulfonic acid disodium salt.A novel Ca(II) coordination polymer, [CaL(H On the basis that metal-organic frameworks can be used as delivery vehicles for drug molecules , 0.0235 and 0.0566; R1, wR2 [I > 2σ(I)], 0.0221 and 0.0557; Rint = 0.0176; goodness of fit on F2, 1.022; largest diff. peak and hole, 0.173 e·Å−3 and −0.213 e·Å−3. Selected bond lengths and angles are listed in A summary of crystal data and relevant refinement parameters for Ca(II) coordination polymer are as following: formula, CSMMC-7721 cells and human lung adenocarcinoma A549 cells were propagated continuously in culture and grown in RPMI 1640 medium with 10% inactivated fetal calf serum and antibiotics. Cell harvested from exponential phase were seeded equivalently into 96 well plates and incubated for 24 h, then the solid compounds studied were added in a concentration gradient. The final concentrations were maintained at c/(μg mL−1) 5, 10, 20, 30, 40, 60 respectively. The plates were maintained at 37 °C in a humidified 5%CO2–90%N2–5%O2 atmosphere and incubated for 48 h, MTT solution was added, and the procedure described in [Human hepatoma ribed in  was then2O)4]n. The spectral properties, crystal structure and antitumor activity also have been investigated. Further investigation of the property and application of Ca(II) coordination polymer are currently in progress in our laboratory.In summary, we have synthesized a new one-dimensional helical chained Ca(II) coordination polymer, [CaL(H


AbstractChelonus (Areselonus) spinigastersp. n., (Hymenoptera: Braconidae: Cheloninae) is described from India. The new species was reared from the moth species Acrocercopslysibathra (Meyr.) on Cordialatifolia Roxb. Lepidoptera or Diptera, PageBreakfollowed to a lesser degree by Coleoptera and Hymenoptera , Ichneumonidae and Braconidae (superfamily Ichneumonoidea) are the most important groups associated with leaf miners.Leaf mining insects are considered as serious pests if leaves of crops are extensively attacked, harvests are reduced or seedling plants even totally destroyed . The majenoptera . Among the world . Parasithe world . ParasitApantelesbambusae Wilkinson and Apantelescalycinae Wilkinson of the leaf miner Cosmopteryxbambusae (Meyrick) and Acrocercopssupplex (Meyrick). Subsequently, Parahormiusjason and Paraharmiusdeiphobus. Chaterjee and Misra (1974) added Epicephalachalybachma (Meyr.) to the host list of Braconlefroyi (Dudgeon and Goughs). Recently, As far as India is concerned, there is a lack of information on braconid parasitoids especially reared from leaf mining lepidopteran hosts. Wilkinson (1928) described two microgastrine parasitoids, viz., Areselonus of the genus Chelonus Panzer (Braconidae: Cheloninae) was named by subgenusAreselonus is given.The subgenus ZDAMU).This study was conducted in the vicinity of western Uttar Pradesh (North India) in order to identify parasitoids of leaf miners along the roadside at AMU campus. The parasitoids were reared in the laboratory in glass rearing jars measuring 8”× 4” at 25 °C +2 with 70 % R.H. in the insectory. The leaves with lepidopteran leaf miners were collected from the plants and transferred to the jars. A complete data set including the date of collection, locality, and name of host plant was maintained. The emerged parasitoids were preserved initially in 75 % alcohol with a few drops of glycerol. These specimens were later mounted on cards. The reared parasitoids were separated based on morphological characters. One braconid species was studied in this research. We followed PageBreakTaxon classificationAnimaliaHymenopteraBraconidaehttp://zoobank.org/E24FBAA6-A4BE-4E6B-9CB7-94AD497FB692Acrocercoplysibathra on Cordialatifolia Roxb. (coll. Shujauddin) . Paratypes, 7♀, 14 ♂♂, with same data as holotype .Holotype, ♀, INDIA: Uttar Pradesh, Aligarh, 11. VIII. 1980., ex. Chelonus (Areselonus) spinigaster sp. n. is closely related to C. (A.) chailini  but differs from it in having the metasoma strongly declivous below the spine  chailini); clypeus rugose (clypeus sparsely punctate in C. (A.) chailini); and the wings hyaline  chailini).Holotype. Female: Body length: 2.4 mm.Head 1.6× as wide as long; eye 1.7× as long as temple in dorsal view; frons strigose, slightly depressed, carina distinct; OOL = 1.5× POL; face rugulose, 1.8× as wide as high, carina absent; clypeus rugose; malar space twice basal width of mandible, latter with two subequal teeth; antenna 16-segmented, subfiliform, extending back slightly beyond base of metasoma, scape twice as long as broad, F1 almost 3.0× as long as wide, this ratio decreases gradually, F8–F11 almost as long as wide, F12–F13 slightly longer than wide and apical segment twice as long as wide.Mesosoma 1.2× as long as wide in lateral view; mesoscutum reticulate-rugose, notauli shallow; scutellum reticulate; propodeum reticulate-rugose, lateral pair of tubercles almost as long as submedian pair.Wings: Fore wing shorter than body; pterostigma twice as long as wide, slightly longer than 1-R1; 3-SR 1.6× as long as r; SR1 curved.PageBreakLegs: Hind femur 3.3× as long as broad, 0.8× as long as hind tibia, hind tibia 1.3× as long wide and 1.3× longer than hind tarsus.Metasoma 2.8× as long as high in lateral view, posteriorly distinctly less than twice as high as basally, strongly convex medially, reticulate-rugose with converging carinae on basal fourth and a small spine at apex; ventral opening not reaching apex, distance from ventral opening to apex of metasoma 1.7× as long as hind basitarsus; ovipositor sheaths in lateral view almost as long as hind basitarsus; metasoma strongly declivous below apical spine .India: Uttar Pradesh.The species name refers to the presence of a spine on the metasoma.PageBreakPageBreak


Linum usitatissimum) hulls using hot water, and its functional groups and antioxidant properties were investigated using infrared spectroscopy and different antioxidant assays , 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid (ABTS), reducing power capacity, and β-carotene bleaching inhibition assay), respectively. The antioxidant capacity of SFG showed interesting DPPH radical-scavenging capacity (IC50 SFG = 2.5 mg·mL−1), strong ABTS radical scavenging activity (% inhibition ABTS = 75.6% ± 2.6% at 40 mg·mL−1), high reducing power capacity (RPSFG = 5 mg·mL−1), and potent β-carotene bleaching inhibition activity (IC50 SFG = 10 mg·mL−1). All of the obtained results demonstrate the promising potential use of SFG in numerous industrial applications, and a way to valorize flaxseed hulls.Soluble flaxseed gum (SFG) was extracted from flax ( Linum usitatissimum L.) was first cultivated as a fiber crop; nonetheless, its value and importance as an oil source has been interestingly increased [−1) compared to other gums, such as locust bean gum, guar gum, and xanthan gum [Flax , DPPH , BHA (butylated hydroxyanisole), Trolox , sodium tetraborate, sodium phosphate, ferric chloride, petroleum ether, and trichloroacetic acid were purchased from Sigma-Aldrich . Ethanol and sulfuric acid were purchased from Scharlab . Ammonium molybdate was obtained from NenTech Ltd. , and potassium ferricyanide was obtained from Loba Chemie . Commercial gum arabic, purchased from a local market , was the hydrocolloid used as reference.Linum usitatissimum) hulls (Flaxseed (m) hulls  of the “Lipid content was determined by Soxhlet method, as previously reported , with slDry matter was determined by drying the hulls at 105 °C in an oven until reaching constant weight, according to the “Association Française de Normalisation” (AFNOR) . Total ng. The recovered SFG was subjected to five washing steps in order to remove the small molecules. The procedure consists of resuspending the SFG in 20 mL water, precipitation by adding 20 mL of ethanol 96%, and centrifugation as described above. Minerals were removed by dialysis (molecular cutoff = 500 Da) using bidistilled water for 3 days. The obtained desalted samples were freeze-dried and stored at 4 °C until analysis.Soluble flaxseed gum (SFG) were extracted from hulls as previously described . In brie−1 after mixing 1 mg from the lyophilized SFG sample with 100 mg KBr. The SFG spectrum was then compared to the FTIR spectrum of gum arabic, in order to check for similarities.SFG functional group analysis was assessed by Fourier transform infrared (FTIR) spectroscopy using Analect Instrument fx-6160  as previously described . The trasample. A control solution without SFG was prepared under the same conditions by mixing 125 μL DPPH solution with 875 μL absolute ethanol, and the absorbance was recorded as Acontrol. A blank solution without DPPH was prepared by mixing SFG (1–20 mg) with 0.5 mL distilled water and 0.5 mL absolute ethanol, and the absorbance was recorded as Ablank. The free radical scavenging activity (% inhibition) was calculated according to Equation (1). The ability of SFG to scavenge DPPH free radicals was evaluated as previously described . In brie•+ radical cations, and the obtained solution was subsequently diluted with distilled water until an absorbance of 0.7 ± 0.02, measured at 734 nm. One milliliter of ABTS•+ diluted solution (A734 nm = 0.7 ± 0.02) was mixed with 10 μL of SFG solution at different concentrations (5 to 40 mg·mL−1), and the reaction mixture was kept at room temperature for 6 min before measuring the absorbance at 734 nm. The assay relies on the ability of the antioxidant molecules to inhibit the oxidation of ABTS radical cation in ABTS•+. SFG scavenging activity  was determined according to Equation (2). 0 represent respectively the absorbance values of the solutions containing or not containing the free radical ABTS•+.The antiradical activity of SFG was also determined against the free radical 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) based on the discoloration of cations, as previously described . A solutThe total antioxidant activity of SFG was evaluated as previously described , with slw/v) prepared in water). The mixtures were kept for 20 min at 50 °C in a thermostatic water bath. One milliliter trichloroacetic acid (10% (w/v) prepared in water) was added to the reaction mixture after cooling to room temperature. All tubes were then centrifuged for 10 min at 3000 g. From each supernatant, 1.5 mL was taken and mixed with 1.5 mL distilled water and 100 μL freshly prepared ferric trichloride solution (0.1% (w/v) prepared in water). After vortexing, the absorbance was measured at 700 nm. A blank solution (without SFG) was prepared under the same conditions.Reducing power capacity of SFG was measured as previously described , with sl0h and A2h, respectively), using Equation (3). 0h (control) and A2h (control) represent the absorbance values of a control solution (0.5 mL bi-distilled water instead of SFG solution) before and after incubation, respectively.The test evaluating β-carotene bleaching by SFG was performed as previously described , with sl® SPSS® Statistics, USA) and was used to determine the significant differences between the results, with a significance level of p < 0.05.Antioxidant activities were performed in triplicate, and results were expressed as average values with standard deviation (SD). Multiple sample comparison of the means was done using analysis of variance (ANOVA)  and compared to the spectrum of gum arabic  value of SFG was also determined, corresponding to 2.5 mg·mL−1. The lowest IC50 value corresponds to the highest DPPH scavenging activity. Results demonstrated that the IC50 value of SFG was lower than that reported for other polysaccharides, such as those extracted from guara fruits (IC50 = 10.8 mg·mL−1) [50 = 10.8 mg·mL−1) [50 = 2.87 mg·mL−1) [50 = 740 μg·mL−1) [50 = 1.61 mg·mL−1) [Antioxidant molecules are able to reduce the stable radical DPPH, having a deep violet color in solution, to the yellow-colored 1,1-diphenyl-2-picrylhydrazyl. The free radical scavenging activity of SFG was assessed by DPPH test a. The obmg·mL−1) , red primg·mL−1) , and almmg·mL−1) , and wasμg·mL−1)  and pistmg·mL−1) . Compare−1, which was lower than that observed for DPPH at the same concentration. The IC50 value of SFG was also determined, corresponding to 26.1 mg·mL−1. However, ABTS scavenging activity remains lower than that observed for Trolox, used as standard.Similarly to DPPH free radical, ABTS showed potential scavenging activity and again demonstrated its ability to reduce free radicals and convert them to more stable products. Results are presented in −1. At this concentration, 10 mg of SFG was equivalent to 461 mg ascorbic acid. Beyond this concentration, TAC continues to increase with lower slope, indicating saturation of the medium. Although the lower antioxidant activity compared to that of ascorbic acid, the obtained results indicate that SFG represents an interesting natural antioxidant molecule.The reduction of phosphomolybdate was followed by the measurement of total antioxidant capacity (TAC). A green complex of phosphate/Mo (V) is formed at acidic pH, which has a maximal absorbance at 695 nm. The TAC of SFG extracted from hulls was determined as described previously, and was expressed as ascorbic acid equivalent (AAE)/mL c. The ob3+ to Fe2+ was followed by measuring the absorbance (A) at 700 nm, corresponding to the formation of Perl’s Prussian blue at 700 nm  [700 nm = 1.74 at 5 mg·mL−1) [700 nm = 3.4 at 20 mg·mL−1) [700 nm = 1.36 at 12 mg·mL−1) [Moreover, the reducing capacity of SFG to convert Fet 700 nm d. The obmg·mL−1) , pistachmg·mL−1) , mushroomg·mL−1) , and fromg·mL−1) .−1. The maximum measured inhibition corresponding to this concentration was 62.4% ± 3.1%, thus showing the efficiency of SFG as a natural antioxidant.Finally, the β-carotene bleaching test simulates the oxidation of lipids’ membrane; thus, it is considered a good model for membrane-based lipid peroxidation. The oxidation of linoleic acid during this assay generates peroxyl free radicals, which are able to oxidize the unsaturated β-carotene. The presence of an antioxidant molecule in the medium will minimize this oxidation. Therefore, the antioxidant activity of SFG could be measured by following the degradation rate of β-carotene. The obtained results d show thIn conclusion, the in vitro antioxidant capacity of soluble flaxseed gum extracted from hulls was demonstrated. Results showed great antioxidant activities through numerous tests, and were compared to commercial standard molecules. SFG presented similar functional groups when compared to gum arabic, which suggests its polysaccharidic structure. These results show the promising potential of flaxseed hulls as a co-product to be fully exploited and extract food additives that could replace other commercial gums, such as gum arabic. However, further studies should be conducted to explain the mechanisms behind these activities.


Yarrowia lipolytica is an increasingly common biofactory. To enhance protein expression, several promoters have been developed, including the constitutive TEF promoter, the inducible POX2 promotor, and the hybrid hp4d promoter. Recently, new hp4d-inspired promoters have been created that couple various numbers of UAS1 tandem elements with the minimal LEU2 promoter or the TEF promoter. Three different protein-secretion signaling sequences can be used: preLip2, preXpr2, and preSuc2.The yeast TEF and hp4d promoters along with five new hybrid promoters: 2UAS1-pTEF, 3UAS1-pTEF, 4UAS1-pTEF, 8UAS1-pTEF, and hp8d. We compared the production of RedStar2, glucoamylase, and xylanase C when strains were grown on three media. As expected, levels of RedStar2 and glucoamylase were greatest in the strain with the 8UAS1-pTEF promoter, which was stronger. However, surprisingly, the 2UAS1-pTEF promoter was associated with the greatest xylanase C production and activity. This finding underscored that stronger promoters are not always better when it comes to protein production. We therefore developed a method for easily identifying the best promoter for a given protein of interest. In this gateway method, genes for YFP and α-amylase were transferred into a pool of vectors containing different promoters and gene expression was then analyzed. We observed that, in most cases, protein production and activity were correlated with promoter strength, although this pattern was protein dependent.To our knowledge, our study is the first to use a set of vectors with promoters of variable strength to produce proteins of industrial interest. We used the more conventional Protein expression depends on more than just promoter strength. Indeed, promoter suitability appears to be protein dependent; in some cases, optimal expression and activity was obtained using a weaker promoter. We showed that using a vector pool containing promoters of variable strength can be a powerful tool for rapidly identifying the best producer for a given protein of interest.The online version of this article (doi:10.1186/s12934-017-0647-3) contains supplementary material, which is available to authorized users. Saccharomyces cerevisiae [Pichia pastoris, Hansanula polymorpha, Kluyveromyces lactis, Kluyveromyces marxianus [Yarrowia lipolytica [Increasing the efficiency of heterologous gene expression is a major goal for the agrifood, bioconversion, and pharmaceutical industries as they have a growing need for recombinant proteins. Expression systems using yeasts present several advantages: yeasts are easy to manipulate, they are unicellular organisms with rapid growth rates, and they are eukaryotes that can incorporate post-translational modifications. In addition to the more conventional revisiae , alternaarxianus –5, and Ypolytica , 7.Y. lipolytica have several advantages [Y. lipolytica is a non-pathogenic organism that can grow on a diversity of substrates. Second, the products of several Y. lipolytica-based processes have received the “generally recognized as safe” (GRAS) designation from the FDA. Third, Y. lipolytica has a naturally strong secretory ability [Production systems exploiting vantages , 8. Firs ability , 8 and d ability .Y. lipolytica. Indeed, integrative expression cassettes containing different markers, such as LEU2, URA3, ADE2, and LYS5, have been constructed. They can be used to transform competent auxotrophic strains of Y. lipolytica. Moreover, several promoters are also available, including the constitutive TEF promoter, the constitutive and hybrid hp4d promoter, and the inducible POX2 and LIP2 promoters [Y. lipolytica to produce such proteins as xylanase, lipase, leucine aminopeptidase II, human interferon, α2b endoglucanase II, and cellobiohydrolase II [Y. lipolytica: preLip2, preXpr2, and preSuc2 [Several genetic tools are available to enhance protein expression in romoters –14. In aromoters –17. Currromoters . All of olase II , 20. Pas preSuc2 –24.Y. lipolytica is better than P. pastoris at producing heterologous proteins [P. pastoris, E. coli, or simple free cells, Y. lipolytica was the best candidate for generating extracellular Lip2 because it can form a stable isotope-labeled version of the protein. Boonvitthya and colleagues [Y. lipolytica and P. pastoris. In YT medium, Y. lipolytica produced up to 15 mg/L of endoglucanase and 50 mg/L of cellobiohydrolase. Furthermore, the enzymes produced by Y. lipolytica had higher levels of specific activity than did their counterparts in P. pastoris. Finally, it has been found that Y. lipolytica has weaker protein glycosylation than does P. pastoris [Several studies have suggested that proteins , 25. Indlleagues  comparedpastoris .TEF1 gene, which encodes the translation elongation factor-1α [XPR2 gene . This discovery led to the development of the hp4d promoter, which is based on the minimal LEU2 promoter and contains four UAS1 tandem elements; with this promoter, expression increases as the number of UAS1 tandem elements increases. More recently, several research groups have used this basic model  to develop improved promoters [One of the first strong constitutive promoters was developed by Novo, using the actor-1α . Later, actor-1α  identifiromoters –29. It hTEF and hp4d, as well as five new hybrid promoters of our own construction. To create the latter, we added two, three, four, or eight UAS1 tandem elements to pTEF; we also added four tandem elements to hp4d. Promoter strength in transformed Y. lipolytica strains was quantified using RedStar2, a fluorescent protein, as a reporter; we also analyzed the production of secreted Aspergillus niger glucoamylase (GA) and xylanase C (XlnC). GA is a glucan 1,4-alpha-glucosidase that belongs to the glycosyl hydrolase family. It catalyzes the degradation of starch and other complex sugars, releasing d-glucose. GA is largely used to produce biolipids and bioethanol from starch or lignocellulosic materials [However, to our knowledge, no study to date has used these UAS1-based promoters to produce proteins of industrial interest. Here, we used two conventional promoters, paterials , 31. XlnOur results revealed that optimal protein expression, secretion, and activity are not always correlated with promoter strength. Consequently, we developed a simple method for improving protein expression that involves the use of a pool of vectors containing promoters of variable strength.Y. lipolytica wild-type strain W29 (ATCC20460) was used as the basis for all the Y. lipolytica strains built in this study  has previously been described by Barth and Gaillardin [Escherichia coli strain DH5α was used to construct the plasmids, except in the case of vectors containing ccdB, for which E. coli strain DB3.1 was used. E. coli growth media and culture conditions have been previously described by Sambrook and colleagues [Y. lipolytica have been described by Barth and Gaillardin [4Cl, and 50 mM phosphate buffer (pH 6.8). This minimal medium was supplemented with uracil (0.1 g/L) and/or leucine (0.1 g/L) as necessary. YP2D4 medium contained 10 g/L of yeast extract , 20 g/L of Bacto Peptone , and 40 g/L of glucose . Solid media were created by adding 1.6% agar.The illardin . Escherilleagues , and thoillardin . Rich meillardin . The YPDI-sceI fragment flanked by LoxP/LoxR [ClaI-BamHI and BamHI-AvrII fragments, respectively. The zeta region for expression cassette integration was flanked by the NotI site, which is involved in the release of the expression cassette prior to transformation. Plasmid and strain construction are described in Additional file BamHI-AvrII , and thClaI3′Hp4d5′ and HYB-BstbI5′Hp4d3′ as primers .The two, three, or four UAS1 tandem element fragments were amplified by PCR using HYB-GA was cloned into the JMP2482, JMP2484, JMP2397, JMP2607, JMP2471, and JMP2473 plasmids at the BamHI and AvrII restriction sites, yielding JMP3781 (LEU2ex 2UAS1-pTEF-GA), JMP3782 (LEU2ex 3UAS1-pTEF-GA), JMP3783 (LEU2ex 4UAS1-pTEF-GA), JMP3784 (LEU2ex 8UAS1-pTEF-GA), JMP3785 (LEU2ex hp4d-GA), and JMP3786 (LEU2ex hp8d-GA), respectively.XlnC was cloned into the JMP2482, JMP2484, JMP2397, JMP2607, JMP2471, and JMP2473 plasmids at the BamHI and AvrII restriction sites, yielding the JMP3096 (LEU2ex 2UAS1-pTEF-XlnC), JMP3097 (LEU2ex 3UAS1-pTEF-XlnC), JMP3098 (LEU2ex 4UAS1-pTEF-XlnC), JMP3099 (LEU2ex 8UAS1-pTEF-XlnC), JMP3100 (LEU2ex hp4d-XlnC), and JMP3101 (LEU2ex hp8d-XlnC) plasmids, respectively.® in accordance with the manufacturer’s instructions using the primers listed in Table The sequences of the genes encoding YFP and α-amylase are provided in Additional file NotI, were used to transform individual strains via the lithium-acetate method [The overexpression cassettes, obtained by digesting the plasmids with e method . Transfoe method . The priRestriction enzymes were obtained from OZYME . PCR was performed using an Eppendorf 2720 thermal cycler; GoTaq DNA polymerases  were employed to verify the results and PyroBest DNA polymerases  were employed to carry out cloning. PCR and DNA fragment purification were performed as previously described . The amo® containing the YFP or α-amylase gene. The transfer of the genes of interest was performed using LR Clonase® in accordance with the manufacturer’s instructions . The mixture was used to transform E. coli strain DB3.1. The resulting transformants were then pooled, and their DNA was extracted and digested before Y. lipolytica was transformed in turn.Forty ng/µL of each of the recipient plasmids was mixed with pENTR™/D-TOPO2D4 media and was concentrated tenfold in 30 mM Tris (pH 8.0) and 50 mM NaCl using Amicon Ultra-0.5 10 K centrifugal filters . Protein production was analyzed via polyacrylamide gel electrophoresis (SDS-PAGE); 4–12% Tris–Glycine gels and an XCell SureLock™ Mini-Cell electrophoresis system  were used. Prism  and wide-range  protein molecular weight markers were used as standards . The gels were stained with 0.2% Coomassie Brilliant Blue R dye .Supernatant was obtained from cultures grown for 72 h in YNB, YPD, or YPTwenty-μL samples were analyzed for protein content using the Coomassie (Bradford) Protein Assay Kit  in accordance with the manufacturer’s instructions.® 3000 system  with an Aminex HPX87H column coupled to an RI detector was used. The column was eluted with 0.01 N H2SO4 at room temperature and a flow rate of 0.6 mL/min. Identification and quantification were achieved via comparison to standards. Enzyme activity was expressed in U mL/L of supernatant, where one unit of GA activity (1 U) was defined as the amount of GA required to release 1 μmol of glucose per minute.GA activity was measured as previously described , with th® Ultra Xylanase Assay Kit  in 30 mM Tris (pH 8.0) and 50 mM NaCl at 25 °C in a BioLector® . Prior to the assays, supernatant from cultures grown in YNB medium was diluted 50- and 100-fold, and supernatant from cultures grown in YPD or YP2D4 was diluted 500- and 1000-fold. As in the case of GA, one unit of XlnC activity (1 U) was defined as the amount of XlnC required to release 1 µmol of xylose per minute.XlnC activity was determined using the EnzChekY. lipolytica strains was analyzed using a microtiter plate reader, as previously described [The growth of the escribed . RedStarImages were acquired using a Zeiss Axio Imager M2 microscope  and Axiovision v. 4.8 software .® Green Supermix Kit . The number of XlnC mRNA copies was determined using the cycle threshold (Ct) values, which were standardized using results for the expression of the actin gene (YALI0D08272g); the number of XlnC mRNA copies found in the strain containing pTEF-XlnC was employed as a reference.RNA extraction was performed using the RNeasy Mini Kit  followed by DNA digestion with DNase I . cDNA synthesis was performed with the Maxima First Strand cDNA Synthesis Kit with dsDNase . PCR quantification was performed with CFX Connect™ Real-Time PCR Detection System  using the SsoAdvanced™ Universal SYBRTEF and hp4d. Four new hybrid promoters were generated by combining two, three, four, or eight UAS1 tandem elements taken from hp4d with the TEF promoter, yielding 2UAS1-pTEF, 3UAS1-pTEF, 4UAS1-pTEF, and 8UAS1-pTEF, respectively  and strongest promoter  . Interestingly, we found that XlnC production was 2–4 times higher in the strain containing 2UAS1-pTEF than in the strains containing pTEF, 8UAS1-pTEF, and hp4d. In our experiment, in YP2D4, maximum XlnC production was about 153 mg/L. The strain containing 8UAS1-pTEF produced slightly more XlnC than the strains containing 3UAS1-pTEF and 4UAS1-pTEF when the yeasts were cultured in YNB. However, its levels of production were similar or lower when the yeasts were cultured in YPD or YP2D4. In various microorganisms, several bottlenecks in heterologous protein production have been identified; they include transcription, protein folding and glycosylation, translocation, signal peptide processing, and proteolysis [TEF was the best promoter for XlnC production. To evaluate if this result could be attributed to the 2UAS1-pTEF promoter resulting in higher levels of XlnC transcription, XlnC mRNA levels were analyzed using qRT-PCR . Derivatives were constructed using ClaI-BamHI-based promoter exchange  and E6 (YFP). Using this method, we have identified several good producers for both enzymes, which shows that it could be very helpful to use a pool of plasmid vectors containing variable-strength promoters to obtain strains that have optimal activity levels.We analyzed the expression of YFP and secreted α-amylase Fig. . Brieflyion Fig. . We founEF Table . Indeed,Y. lipolytica; however, these promoters were only used to produce intracellular proteins, such as fluorescent proteins, or in β-galactosidase assays. We constructed similar versions of these promoters  and analyzed their impact on the production of intracellular proteins, namely RedStar2 and YFP, as well as extracellular proteins, namely glucoamylase, xylanase C, and α-amylase (see summary in Table TEF) resulted in the highest levels of protein production and activity . However, the best promoters for xylanase C were 2UAS1-pTEF and hp8d. Our results show that stronger promoters do not always optimize protein production and activity. It could be that either transcriptional or post-translational regulation, such as RNA processing and stability, translation efficiency, or protein stability and modification [®. Indeed, in a single step, it was possible to obtain a collection of vectors containing variable-strength promoters upstream from the gene of interest. Once the study organism has been transformed, screening tests can be used to select the best strains. This approach could be very helpful to those seeking to improve protein production, whether in a research or an industrial setting. The pool should contain a decent number of promoters and include inducible promoters, which could be particularly important when dealing with toxic proteins.Blazeck and colleagues , 28 devefication , 46, pla


Mammalian mitochondrial genome encodes a small set of tRNAs, rRNAs, and mRNAs. The RNA synthesis process has been well characterized. How the RNAs are degraded, however, is poorly understood. It was long assumed that the degradation happens in the matrix where transcription and translation machineries reside. Here we show that contrary to the assumption, mammalian mitochondrial RNA degradation occurs in the mitochondrial intermembrane space (IMS) and the IMS-localized RNASET2 is the enzyme that degrades the RNAs. This provides a new paradigm for understanding mitochondrial RNA metabolism and transport.The online version of this article (doi:10.1007/s13238-017-0448-9) contains supplementary material, which is available to authorized users. Mammalian mitochondria contain a small circular genome, which encodes 2 ribosomal RNAs, 22 tRNAs, and 13 essential protein subunits in the OXPHOS pathway . Degradation of the newly synthesized mtRNAs was examined under various chasing conditions. Slower degradation was observed at 25°C than at 37°C   but an inhibitory effect at a higher concentration (20 mmol/L)  but inhibitory at a higher concentration (8 mmol/L) . Based on the mass-spectrometry results of the purified samples (Tables S1 and S2), we singled out RNASET2 for further testing. Mammalian RNASET2s are endoribonucleases that are targeted for secretion and also to lysosomes , RNASET2 overexpression led to a significant decrease of the mtRNA levels  without the N-terminal 24 amino acids to the targeting sequence of cytochrome C1 that targeted the protein to the IMS (C1ΔNT2M). Unlike the wild-type RNASET2, C1ΔNT2M localizes predominately in the mitochondria , suggesting the activity of human RNASET2 is intrinsically different from its protozoan counterparts.The response of purified RNASET2 to pH is of great interest. Protozoan RNASET2s are mostly acidic ribonucleases Irie, , but theE. coli . Unlike RNASET2, Endo G was sensitive to proteinase K and the responses to the conditions tested were vastly different from those of RNASET2 or in organello mitochondrial RNA degradation. Thus, a direct involvement of Endo G on mitochondrial RNA degradation seems unlikely. That the human RNASET2 purified from mammalian cells and bacteria showed responses to so many conditions almost identical to those of the in organello mtRNA degradation together with its IMS localization further verified it as the enzyme responsible for mtRNA degradation.Another IMS localized nuclease is Endo G  had been a great mystery. Our attempt at unraveling this mystery uncovered a mitochondrial IMS-localized ribonuclease activity that degrades mtRNAs and is sensitive to many cellular conditions such as pH and CaLeishmania tarentolae mitochondria -HAHisPC, PQCXIP-RNASET2-GFP, PQCXIP-RNASET2 (C184R)-GFP, PET28A-RNASET2-HisPC, PQCXIP-EndoG-HAHis, PET28A-EndoG-HAHis, PQCXIP-APEX-HisPC, PQCXIP-RNASET2-APEX-HisPC, PQCXIP-MIA40-APEX-HisPC, PQCXIP-PNPASE-HAHis, PQCXIP-MIA40-Avi-HisFlag, PQCXIP-RNASET2-Avi-HisFlag, and PQCXIP-TOM22-BirA-HAHis. See Supplemental Experimental Procedures for construction details.Plasmids used include g for 5 min, and the homogenization repeated once. Supernatants from both times were centrifuged again at 700 ×g. Crude mitochondria were pelleted from second-round supernatants at 11,000 ×g for 5 min, washed once with mitoprep buffer and resuspended in mitoprep buffer of desired volume for further use. Post-mitochondrial supernatant was spun at 21,000 ×g for 10 min and the supernatant was collected as cytosol.Cells were washed once with PBS buffer, resuspended in ice-cold mitoprep buffer , and broken in a glass-Teflon homogenizer on ice with 30 strokes. Nuclei and unbroken cells were pelleted at 700 ×g for 4 min. Salt wash was performed by adding 300 mmol/L of NaCl (from 5 mol/L stock) into mitoprep buffer (in cases of intact mitochondria) or mitoplasting mixture (in cases of mitoplasts) after 20 min on ice for another 5 min. Mitochondrial matrix was isolated by sonicating the salt-washed mitoplast in mitoprep buffer and separating the soluble fraction (matrix) from membrane by 10 min 21,000 ×g centrifugation. Total soluble was isolated by sonicating mitochondria in mitoprep buffer and separating the soluble fraction  from membranes by 10 min 21,000 ×g centrifugation. The pellet was washed twice with mitoprep buffer and used as total membranes.Hypotonic treatment was performed by incubating mitochondria for 20 min on ice by diluting mitoprep buffer with 10 volumes of 20 mmol/L HEPES pH 7.4 with one gentle vortexing at 10 min. Mitoplasts were separated from IMS by centrifugation at 15,000 ×g 4 min spin, washed with 1 mL mitoprep buffer, and resuspended in 300 μL mitoprep buffer with 2 μg proteinase K. The mixture was incubated on ice for 15 min with one vortexing at 8 min. 1 mL mitoprep buffer was added after the incubation and mitoplasts were pelleted at 15,000 ×g for 4 min, washed once with 1 mL mitoprep buffer, and proceeded to in organello degradation. Control mitochondria samples skipped the last two spins, as spinning and resuspending at this stage ruptures the outer membrane. This new method was used to ensure degradation of some inner membrane bound proteins that might be involved in mtRNA export from the matrix.Mitoplasting after in organello mtRNA synthesis was carried out by proteinase K treatment. 500 μg mitochondria in 200 μL in organello synthesis mixture were pelleted by 12,000 ×In vitro degradation assay for PNPASE was performed as previously described  and incubating at 90°C for 5 min. Samples were cooled to room temperature and 0.5 μg of proteinase K was added for a 5 min incubation at 37°C. Biotinylated samples were run on SDS-PAGE, transferred to a nylon membrane, and detected with nucleic acid detection kit (Thermo). mtRNA samples were run either on SDS-PAGE or agarose gels and stained with EtBr.2 and 1 μL Biotin RNA Labeling Mix (Roche) with 500 μg mitochondria at 37°C. For each time point , 60 μL reaction mix was taken out and mitochondria were pelleted at 18,000 ×g for 2 min. Pellets were stored at −80°C for at least 15 min before next preparation step. For loading, samples were taken out of −80°C, quickly dissolved in 30 μL SDS-Urea-EDTA buffer (SDS loading buffer with 8 mol/L urea and 15 mmol/L EDTA) preheated to 90°C, and incubated at 90°C for 5 min. Samples were then cooled to room temperature and 0.5 μg of proteinase K was added for a 5 min incubation at 37°C. Biotinylated samples were run on SDS-PAGE, transferred to a nylon membrane (400 mA for 1.5 h), and detected with nucleic acid detection kit (Thermo). These synthesis conditions are less inhibitory to mtRNA degradation, and were used for the in organello degradation assays that examined their responses to mitoplasting, temperature, pH, ATP, and metal ions.In organello RNA synthesis was performed in 200 μL mitoprep buffer containing 4 mmol/L ATP pH 7.4, 20 mmol/L succinate, 1 mmol/L CaCl2, 10 mmol/L MgCl2, and 1 mg/mL HEK cytosol.For comparison of in organello synthesis between different cell lines, more inhibitory synthesis conditions were used to ensure the yield of newly synthesized RNA more representing the real synthesis rate. Therefore, in organello mtRNA synthesis were carried out in buffer containing 4 mmol/L ATP pH 7.4, 20 mmol/L succinate, 0.5 mmol/L CaCl2, and 1 μL Biotin RNA Labeling Mix (Roche) at 37°C for 45 min. Mitochondria were pelleted at 12,000 ×g for 4 min at 4°C, washed with 1 mL ice cold mitoprep buffer, resuspended in 150 μL mitoprep buffer, and incubated on ice for 15 min with one vortexing at 8 min. 50 μL ice cold cocktail containing 4 mmol/L UTP, 40 mmol/L Ca2+, 1 μL Ribolock RNase inhibitor (Thermo), and 1 μg RNaseI (Thermo)  in mitoprep buffer was added to the sample (RNase inhibitor was used to eliminate cytosolic interference and RNaseI was used to quickly degrade the RNA from broken mitochondria). The samples were shifted to 37°C. For each time point , 60 μL reaction mix was taken out and mitochondria were pelleted at 18,000 ×g for 2 min. Pellets were stored at −80°C for at least 15 min before next preparation step. For loading, samples were taken out of −80°C, quickly dissolved in 30 μL SDS-Urea-EDTA buffer (SDS loading buffer with 8 M urea and 15 mmol/L EDTA) preheated to 90°C, and incubated at 90°C for 5 min. Samples were then cooled to room temperature and 0.5 μg of proteinase K was added for a 5 min incubation at 37°C. Biotinylated samples were run on SDS-PAGE, transferred to a nylon membrane (400 mA for 1.5 h), and detected with nucleic acid detection kit (Thermo).For in organello degradation assays that examined their responses to mitoplasting, temperature, pH, ATP, and metal ions, 500 μg mitochondria first underwent mtRNA synthesis in 200 μL mitoprep buffer containing 4 mmol/L ATP pH 7.4, 20 mmol/L succinate, 1 mmol/L CaCl2, and 1 mg/mL HEK cytosol for 45 min at 37°C before the following wash and degradation steps. Mitochondria should be isolated from stable cells lines grown to less than 5 doubling times from the initial frozen stocks.For comparison of in organello mtRNA degradation between different cell lines, in organello mtRNA synthesis were carried out in buffer containing 4 mmol/L ATP pH 7.4, 20 mmol/L succinate, 0.5 mmol/L CaCl2O2 was added and the plates were gently agitated for 1 min. The reaction was quickly quenched by replacing the medium with DPBS containing 5 mmol/L Trolox (abcam) and 10 mmol/L sodium ascorbate (Solarbio). Cells were then washed with the same solution three times. For Ni-NTA (Qigen) and HA bead (Thermo Scientific) enrichment of PNPASE, mitochondria were isolated from the biotin-labeled cells and PNPASE was first purified from the lysate under denaturing condition using Ni-NTA beads and then using HA beads under native conditions according to the manufactures’ instructions.500 μmol/L biotin-phenol (ApexBio) was added to cell medium for 30 min. Then 1 mmol/L of HHEK293 cells co-transfected with the Avi-tagged protein expressing plasmid and BirA fusion protein expressing plasmid were gown in medium containing 25 μmol/L D-Biotin (Amresco) for 12 h. The cells were harvested, washed with PBS time times, lysed in 1× SDS protein loading buffer, and run on SDS-PAGE for biotin detection.in vitro transcription. See Supplemental Experimental Procedures for details.Additional procedures include protein purification, Western blotting, mtRNA isolation, qPCR, Optiprep gradient centrifugation, identification of IMS ribonuclease, fluorescence microscopy and image acquisition, and Supplementary material 1 (PDF 1633 kb)Below is the link to the electronic supplementary material.


Campylobacter jejuni CAM970 and C. coli CAM962, which were associated with a large outbreak of foodborne illness originating from undercooked chicken sushi in Fukuoka, Japan, in May 2016. Their genome sizes were 1,690,901 and 1,704,736 bp, with 22 and 23 rRNAs, 9 and 9 tRNAs, and 411× and 419× coverage for C. jejuni CAM970 and C. coli CAM962, respectively.Here, we report the draft genome sequences of  Campylobacter jejuni and C. coli are two of the leading causes of foodborne gastrointestinal illnesses, which, worldwide, number approximately 400 to 500 million cases annually  in Fukuoka city. Bacteriological surveillance determined that both C. jejuni and C. coli were associated with the outbreak. In order to characterize their genomic traits, we examined, and report here, the draft genome sequences of C. jejuni CAM970 and C. coli CAM962, which were isolated from patients treated during the outbreak. Genomic DNA of the two strains was sequenced by single-end sequencing with an Ion Torrent PGM sequencer , resulting in average coverages of 411× and 419×, respectively. Raw reads were trimmed and de novo assembled using CLC Genomics Workbench version 9.0 . The parameters for trimming were as follows: ambiguous limit, 2; quality limit, 0.05; number of 5′ terminal nucleotides, 20; and number of 3′ terminal nucleotides, 5. The parameters for the de novo assembly were as follows: mapping mode, create simple contig sequences (fast); bubble size, 50; word size, 21; minimum contig length, 500 bp; perform scaffolding, no; and autodetect paired distances, yes. The draft genomes of C. jejuni CAM970 and C. coli CAM962 were assembled into 61 and 34 contigs with accumulated lengths of 1,690,901 and 1,704,736 bp  and average G+C contents of 30.2 or 31.3%, respectively. The genome was annotated with the RAST server . The data provided here can aid in future efforts to identify the sources of infections, since the genome sequence-based framework has potential advantages for epidemiological studies of Campylobacter infections  (Campylobacter spp. associated with foodborne outbreaks will contribute to improving our understanding of host and geographic adaptation of this pathogen and lead to the development of preventive strategies.The sequence types (STs) of these strains were also profiled with a multilocus sequence typing (MLST) scheme . The resfections . With thracking) , 9, furtBDRZ01000000 (CAM970) and BDRY01000000 (CAM962). The versions described in this paper are the first versions, BDRZ01000000 (CAM970) and BDRY01000000 (CAM962).This whole-genome shotgun project has been deposited at DDBJ/EMBL/GenBank under the accession numbers