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Plaque assays were conducted using a L2 fibroblast cell line as previously described (32) with minor modifications for visualization and quantification of plaques. L2 fibroblasts were seeded at 1.2 × 106 per 35-mm-diameter dish and infected at an MOI of 0.5 to obtain approximately 30 PFU per dish. At 4 to 6 days postinfection, cells were stained with 0.3% crystal violet for 10 min and washed twice with deionized water. Stained wells were imaged and areas of plaque formation were measured on Fiji image analysis software (68).
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| 100.0 |
Female C57BL/6 mice (6 to 8 weeks of age) were injected intravenously with 1 × 105 CFU (50% lethal dose [LD50] = 1 for wild-type 10403s L. monocytogenes) of logarithmically growing L. monocytogenes (optical density at 600 nm [OD600] = 0.5). At 48 h postinfection, spleens and livers were harvested, homogenized, and enumerated for CFU as previously described (12).
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| 100.0 |
L. monocytogenes strains were grown to mid-late-logarithmic phase in BHI media at 37°C. Bacteria were diluted in phosphate-buffered saline (PBS) to a concentration of 106/ml and transferred to a fluorescence-activated cell sorter (FACS) tube. Next, cells were stained with 3 mM membrane potential indicator dye DIO2(3) (3,3′-diethyloxacarbocyanine iodide) (Sigma; 320684) and/or 500 µM proton ionophore CCCP (carbonyl cyanide 3-chlorophenylhydrazone) (Sigma; C2759) for 30 min. Samples were analyzed on a BD LSR-II flow cytometer, and data analysis was performed using FlowJo software. Samples were gated for bacteria using forward and side scatter, and then individual bacteria were measured for ratiometric fluorescence analysis using comparisons between red mean fluorescence intensity and green mean fluorescence intensity.
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| 99.94 |
L. monocytogenes strains were grown in aerated BHI cultures with various concentrations of ceftriaxone (Sigma; C5793), ampicillin (Sigma; A0166), bacitracin (Fisher Bioreagent; BP29501), daptomycin (Merck), lysozyme (Sigma; L6876), LL-37 (AnaSpec; AS61302), or hydrogen peroxide. MICs were determined as the median concentration of antimicrobial required to prevent replication over 12 h as determined by OD600 in a BioTek Eon or BioTek Synergy HT plate reader.
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| 99.94 |
iIFNAR−/− macrophages were grown in 35 mm-diameter dishes at a density of 2 × 106 per dish. Cells were infected with L. monocytogenes at an MOI of 10 for 6 h or treated for 1 h with 100 µM menadione (positive control). N-acetylcysteine (NAC) (1 mM) was added to cell cultures throughout treatment to scavenge ROS. Treated cells were stained with 2.5 µM CellROX DeepRed (Molecular Probes; C10422) for 30 min at 37°C. Cells were then washed twice in FACS buffer, fixed with 3.7% formaldehyde for 15 min, and washed again prior to analysis on a BD LSR-II flow cytometer.
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| 99.94 |
Statistical significance analysis (GraphPad Prism 6.0h) was performed using one-way or two-way analysis of variance (ANOVA) with the Bonferroni posttest unless otherwise indicated in the figure legends. Bacteriolysis assay data were log transformed prior to statistical analysis (*, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001).
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| 99.94 |
Bacteria from the Marinitoga genus are thermophilic, anaerobic, and organotrophic microorganisms isolated from various hot environments such as deep-sea hydrothermal vents or costal thermal springs. Among the five Marinitoga species described (1–5), only the M. piezophila KA3 genome has been sequenced (6). Here, we present the draft genome sequences of two novel Marinitoga camini isolates, DV1155 and DV1197, both containing proviral sequences.
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| 100.0 |
The two strains were sampled during the DIVA 2 cruise in 1994 (7). M. camini DV1155 was isolated from a black smoker chimney at the Menez Gwen site. This hydrothermal field is located on the Mid-Atlantic Ridge at a depth of 840 to 870 m (8). M. camini DV1197 was isolated from a colonization module deployed at the Lucky strike hydrothermal vent field. This site is located at 1700 m of depth in the Mid-Atlantic Ridge (8). Strains DV1197 and DV1155 grow optimally at 60°C and 65°C, respectively, at atmospheric pressure in a modified Ravot medium (9) with elemental sulfur instead of cysteine.
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| 99.94 |
Genomic DNA was extracted following the protocol of Geslin et al. (10). The purity and quantity of the DNA were measured using Nanodrop and Qubit instruments (Thermo, Fisher Scientific). Shotgun libraries were constructed using the Nextera XT kit and sequenced as one of ten pooled, barcoded libraries on a MiSeq (all from Illumina) using 500 cycles generating 2 × 250 bp paired-end reads. The genomes assembled de novo by CLC Genomics Workbench 7.0.4, using trimming default settings, automatic word size, a bubble size corresponding to the average length of the input reads, a minimum contig length of 1000 bp, and reads mapped back to the contigs.
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| 99.94 |
For M. camini DV1155 this resulted in 56 contigs totaling 2,435,399 bp, with an N50 of 90,885 bp, longest contig size of 254.99 bp, and G+C content of 27.3%. No extrachromosomal DNA was observed but a proviral sequence of 50,700 pb was found in the genome using Prophinder (11). For M. camini DV1197, we obtained 51 contigs totaling 2,274,557 bp with an N50 of 72,669, longest contig size of 188,990 bp, and a G+C content of 27.4%. No extrachromosomal DNA was observed but a proviral sequence of 53,437 bp was detected using the same methods than for M. camini DV1155.
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| 100.0 |
Both whole-genome shotgun projects have been deposited at DDBJ/EMBL/GenBank under the accession no. AZAX00000000 and AZAY00000000 for M. camini DV1155 and M. camini DV1197, respectively. The versions described in this paper are the first versions, AZAX01000000 and AZAY01000000.
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| 99.94 |
Terpenoids, also known as isoprenoids, are one of the largest and most diverse classes of naturally occurring products . In animals, terpenoids play crucial roles as membrane constituents (e.g., cholesterol) and components of the respiratory electron transport chain (e.g., ubiquinone) , whereas in microbes and plants, they are found as secondary metabolites that have been used as pharmaceuticals (e.g., paclitaxel and artemisinin), flavors, and fragrances (e.g., menthol and patchoulol) . Terpenoids, including monoterpenes, sesquiterpenes, and diterpenes, are synthesized by terpene synthases (TPSs) with universal five-carbon building units, isopentenyl diphosphate (IPP) and its isomer dimethyl allyldiphosphate (DMAPP). IPP and DMAPP are synthesized in two different pathways: the mevalonate (MVA) pathway , and the 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway (Fig. 1a). In general, Gram-negative bacteria, including Escherichia coli, employ the MEP pathway, whereas eukaryotes, archaea, humans, and Gram-positive cocci use the MVA pathway . Prenyltransferases assemble the IPP and DMAPP units to produce the prenyl diphosphates, which are terpenoid precursors, including geranyl diphosphate (GPP, C10), farnesyl diphosphate (FPP, C15), and geranyl geranyl diphosphate (GGPP, C20). These prenyl diphosphate molecules are converted into a large variety of terpenoids by TPSs [7, 8].Fig. 1Biosynthesis of (−)-α-bisabolol in engineered E. coli. a Schematic representation of (−)-α-bisabolol production in an engineered E. coli harboring endogenous 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway genes and exogenous mevalonate (MVA) pathway genes. The native MEP pathway consists of: DXS deoxyxylulose 5-phosphate synthase; DXR deoxyxylulose 5-phosphate reductoisomerase; CMS 2-C-methylerythritol 4-phosphate cytidyl transferase; CMK 4-(cytidine 5′-diphospho)-2-C-methylerythritol kinase; MDS 2-C-methylerythritol 2,4-cyclodiphosphate synthase; HDS (E)-4-hydroxy-3-methylbut-2-enyl diphosphate reductase; and HDR hydroxymethylbutenyl diphosphate reductase. The engineered mevalonate pathway consists of seven enzymes: MvaE, acetoacetyl-CoA thiolase (GenBank accession number: AF290092); MvaS, 3-hydroxy-3-methylglutaryl- CoA synthase (GenBank accession number: AF290092); MvaA, 3-hydroxy-3-methylglutaryl-CoA reductase (GenBank accession number: AF290092); MvaK1, mevalonate kinase (GenBank accession number: AF290099); MvaK2, phosphomevalonate kinase (GenBank accession number: AF290099); MvaD, mevalonate 5-diphosphate decarboxylase (GenBank accession number: AF290099); Idi, isopentenyl diphosphate isomerase (GenBank accession number: AF119715); and IspA, geranyl diphosphate synthase or farnesyl diphosphate synthase (GenBank accession number: AAC73524). MrBBS; E. coli codon-optimized (−)-α-bisabolol synthase of German chamomile (GenBank accession number: KU680479). b Plasmid constructs used for (−)-α-bisabolol production in E. coli. pTSN-MrBBS, pTrc99A with MrBBS gene from German chamomile; pSSN12Didi, pSTV28 containing mvaK1, mvaD, mvaK2 of Streptococcus pneumoniae, and idi of Escherichia coli; pSNA-MrBBS, pSTV28 containing mvaK1, mvaK2, and mvaD of S. pneumoniae, idi of E. coli, mvaE and mvaS of Enterococcus faecalis, and MrBBS of German chamomile; pSNA-MrBBS-IspA, pSNA-MrBBS containing ispA of E. coli
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study
| 99.56 |
Biosynthesis of (−)-α-bisabolol in engineered E. coli. a Schematic representation of (−)-α-bisabolol production in an engineered E. coli harboring endogenous 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway genes and exogenous mevalonate (MVA) pathway genes. The native MEP pathway consists of: DXS deoxyxylulose 5-phosphate synthase; DXR deoxyxylulose 5-phosphate reductoisomerase; CMS 2-C-methylerythritol 4-phosphate cytidyl transferase; CMK 4-(cytidine 5′-diphospho)-2-C-methylerythritol kinase; MDS 2-C-methylerythritol 2,4-cyclodiphosphate synthase; HDS (E)-4-hydroxy-3-methylbut-2-enyl diphosphate reductase; and HDR hydroxymethylbutenyl diphosphate reductase. The engineered mevalonate pathway consists of seven enzymes: MvaE, acetoacetyl-CoA thiolase (GenBank accession number: AF290092); MvaS, 3-hydroxy-3-methylglutaryl- CoA synthase (GenBank accession number: AF290092); MvaA, 3-hydroxy-3-methylglutaryl-CoA reductase (GenBank accession number: AF290092); MvaK1, mevalonate kinase (GenBank accession number: AF290099); MvaK2, phosphomevalonate kinase (GenBank accession number: AF290099); MvaD, mevalonate 5-diphosphate decarboxylase (GenBank accession number: AF290099); Idi, isopentenyl diphosphate isomerase (GenBank accession number: AF119715); and IspA, geranyl diphosphate synthase or farnesyl diphosphate synthase (GenBank accession number: AAC73524). MrBBS; E. coli codon-optimized (−)-α-bisabolol synthase of German chamomile (GenBank accession number: KU680479). b Plasmid constructs used for (−)-α-bisabolol production in E. coli. pTSN-MrBBS, pTrc99A with MrBBS gene from German chamomile; pSSN12Didi, pSTV28 containing mvaK1, mvaD, mvaK2 of Streptococcus pneumoniae, and idi of Escherichia coli; pSNA-MrBBS, pSTV28 containing mvaK1, mvaK2, and mvaD of S. pneumoniae, idi of E. coli, mvaE and mvaS of Enterococcus faecalis, and MrBBS of German chamomile; pSNA-MrBBS-IspA, pSNA-MrBBS containing ispA of E. coli
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study
| 99.94 |
(−)-α-Bisabolol is an unsaturated sesquiterpene alcohol that occurs naturally in the Brazilian candeia tree (Eremanthus erythropappus) and in medicinal herbs such as German chamomile (Matricaria recutita) [9, 10]. It has been shown to have pharmaceutical functions (e.g., antibacterial, antiseptic, and anti-inflammatory activities), and skin-soothing and -moisturizing properties [11–13]. Owing to the low toxicity associated with (−)-α-bisabolol, the Food and Drug Administration (FDA) has granted it the Generally Regarded as Safe (GRAS) status that has promoted its use as an active ingredient in several commercial products . At present, natural (−)-α-bisabolol is primarily manufactured through the steam-distillation of candeia essential oils extracted from the Brazilian candeia tree, which has raised environmental and bioconservation issues in recent years . (−)-α-Bisabolol can also be chemically synthesized; however, chemical synthesis requires an additional economically unviable purification step because of the formation of other diastereomers ((+)-α-bisabolol and (±)-epi-α-bisabolol) and undesirable byproducts . Therefore, a sustainable supply of nature-identical (−)-α-bisabolol is essential to specialty chemical industries. Recently, a (−)-α-bisabolol synthase capable of synthesizing enantio-selective (−)-α-bisabolol as a single terpenoid product has been identified in German chamomile. (−)-α-bisabolol synthase was expressed in Saccharomyces cerevisiae for the production of (−)-α-bisabolol and had a yield of 8 mg/L after 4 days of cultivation . This previous study has paved the way for the biological production of enantioselective (−)-α-bisabolol in engineered microorganisms, but the productivity is very low for industrial production of (−)-α-bisabolol.
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study
| 99.94 |
The aim of this study was to produce (−)-α-bisabolol by large-scale fermentation of E. coli, which has been widely used for various industrial applications, and to develop an economically viable direct (−)-α-bisabolol extraction process during fermentation. To achieve this, we created (−)-α-bisabolol-producing E. coli in three steps: (1) We introduced the German chamomile (−)-α-bisabolol synthase gene (MrBBS) into E. coli convert endogenous FPP to (−)-α-bisabolol; (2) We engineered the exogenous MVA pathway to increase IPP and DMAPP pool; and (3) We overexpressed an ispA gene encoding an FPP synthase that efficiently provides the (−)-α-bisabolol precursor (FPP) from IPP and DMAPP (Fig. 1b).
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study
| 100.0 |
Strains used in this study are listed in Table 1. E. coli DH5α was used for cloning experiments and (−)-α-bisabolol production. BL21(DE3) and MG1655 strains were used to compare effects of different host strains on the production and toxicity of (−)-α-bisabolol. E. coli strains were grown in Luria Bertani (LB) medium (10 g/L tryptone, 5 g/L yeast extract, and 5 g/L NaCl) for cloning experiments and (−)-α-bisabolol production. To produce (−)-α-bisabolol in E. coli, we used various media at 30 °C and 200 rpm; terrific broth (TB) medium (12 g/L enzymatic casein digest, 24 g/L yeast extract, 9.4 g/L K2HPO4, and 2.2 g/L KH2PO4), 2xYT medium (16 g/L tryptone, 10 g/L yeast extract, and 5 g/L NaCl), and M9 minimal medium (6.78 g/L Na2HPO4, 3 g/L KH2PO4, 0.5 g/L NaCl, 1 g/L NH4Cl, 0.241 g/L MgSO4, 0.0111 g/L CaCl2, and 0.1 g/L thiamine). M9 minimal medium was supplemented with 4 g/L of glucose. Ampicillin (100 μg/mL), chloramphenicol (34 μg/mL), or isopropyl β-d-1-thiogalactopyranoside (IPTG) as required.Table 1List of strains, plasmids, and primers used in this studyNameDescriptionRefsStrains DH5α F − , Φ80lacZ·ΔM15·ƒ(lacZYA−argF)U169 deoR recA1 endA1 hsdR17(rk−, mk+) phoA supE44 thi-1 gyrA96 relA1 Enzynomics MG1655F−, λ−, ilvG −, rfb-50, rph1ATCC 700926 BL21(DE3)BL21 F−, dcm ompT hsdS (rB- mB-) gal λ(DE3)EnzynomicsPlasmids pSSN12DidipSTV28 containing mvaK1, mvaD, mvaK2 of Streptococcus pneumoniae, and idi of E. coli pPROLar.APlac/ara-1 expression vector, Kanr, p15A oriClontech pTrc99APtrc expression vector, Ampr, lacIq, pBR322 oriGE Healthcare pTSN-MrBBSpTrc99A containing MrBBS of Matricaria recutita This study pPR-IspApPROLar.A containing ispA of E. coli This study pTSN-MrBBS-IspApTSN-MrBBS containing ispA of E. coli This study pSNApSTV28 containing mvaK1, mvaK2, and mvaD of S. pneumoniae, idi of E. coli, and mvaE and mvaS, of Enterococcus faecalis pSNA-MrBBSpSNA containing MrBBS of M. recutita This study pSNA-MrBBS-IspApSNA-MrBBS containing ispA of E. coli This studyPrimersa Bis-IF aggttaaaccatgagcacactgagcgtcagThis study Bis-IR cgactctagattagactatcatcggatgtaThis study Bis-VF gatagtctaatctagagtcgacctgcaggcThis study Bis-VR gtgtgctcatggtttaacctcctgtgtgaaattgttatcThis study IspAI-F ggtaccccatatggactttccgcagcaactThis study IspAI-R tgcctctagattatttattacgctggatgaThis study IspAV-F taataaataatctagaggcatcaaataaaaThis study IspAV-R gaaagtccatatggggtacctttctcctctThis study IspAop-IF acatccgatgatagtctaatattcattaaagaggagaaagThis study IspAop-IR tgcatgcctgcaggtcgactctagattatttattacgctgThis studyOverlap region for Gibson Assembly is underlined aPrimer sequences are indicated in the 5′–3′ direction
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| 100.0 |
n-Dodecane was used as an overlay to prevent the loss of volatiles. Furthermore, n-dodecane was used to solubilize and extract (−)-α-bisabolol, which is toxic to E. coli cell growth in high concentrations, from the culture media during cultivation. MVA was prepared from mevalonolactone (Sigma Aldrich) as previously described . Cell growth was monitored by measuring the optical density at a wavelength of 600 nm (OD600) with a spectrophotometer (Ultrospec 8000, GE Healthcare, Uppsala, Sweden). The inhibitory effect of (−)-α-bisabolol on the growth of E. coli strains was investigated in LB liquid medium supplemented with various concentrations of (−)-α-bisabolol. Diluted samples from cultures during the stationary phase were plated on LB solid medium and the colony-forming units (CFUs) were determined after overnight incubation at 30 °C.
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study
| 100.0 |
Plasmids and polymerase chain reaction (PCR) primers used in this study are listed in Table 1. Common procedures, including genomic DNA preparation, restriction digestions, transformations, and other standard molecular biological techniques were performed as previously described . All restriction enzymes and T4 DNA ligase were purchased from New England Biolabs (Ipswich, USA). PCR was performed following the manufacturer’s instructions, using a high fidelity KOD-Plus-Neo polymerase (Toyobo, Osaka, Japan). Plasmid preparation and gel extraction kits were obtained from Promega (Madison, USA) and oligonucleotides were synthesized by Bioneer (Daejeon, Korea). The MrBBS gene (GenBank accession number: KJ020282) was optimized to E. coli codons and synthesized by Bioneer (Additional file 1: Figure S1). We deposited the nucleotide sequence data of E. coli codon-optimized MrBBS in the GenBank database (accession number: KU680479). The synthesized MrBBS was amplified by PCR with Bis-IF and Bis-IR primers (Table 1) and the plasmid backbone was amplified with the Bis-VF and Bis-VR primers from pTrc99A (GE Healthcare). The two PCR-amplicons were assembled via the Gibson Assembly Method using Gibson Assembly Master Mix (New England Biolabs), to produce the pTSN-MrBBS plasmid. The entire polycistronic MVA pathway genes were inserted into pTSN-MrBBS through XbaI restriction site digestion of both the pTSN-MrBBS and the pSNA plasmids containing all the mevalonate pathway genes , followed by ligation with T4 DNA ligase. The resultant plasmid was designated pSNA-MrBBS. To overexpress FPP synthase, the ispA gene (GenBank accession number: AAC73524) was PCR-amplified from E. coli MG1655 genomic DNA, using IspAI-F and IspAI-R primers. To avoid proofreading errors introduced during PCR, the amplified ispA gene was first inserted into the XbaI restriction site of pTSN-MrBBS (instead of pSNA-MrBBS). The pTSN-MrBBS-IspA and pSNA plasmids were digested with XbaI followed by ligation with T4 DNA ligase to create pSNA-MrBBS-IspA. The direction of the MVA pathway genes was verified by Sanger sequencing.
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To prepare the pre-culture, the engineered E. coli was cultured in 100 mL of TB medium at 30 °C and 180 rpm. The batch fermentation was performed by inoculating 1% (v/v) of pre-culture into 400 mL TB medium supplemented with 10 g/L glycerol and overlaid with 80 mL n-dodecane in a 1 L fermenter. The cultures were incubated at 30 °C and air-aerated with a flow rate of 1 volume of air per volume of medium per min (vvm), the agitation speed remained fixed at 200 rpm in all experiments. Glycerol was used as a carbon source at a concentration of 10 g/L. The pH was balanced to pH 7.0 by the addition of 1 M HCl and 25% NH4OH solutions. n-Dodecane was overlaid up to 20% (v/v) of culture volume to continuously extract (−)-α-bisabolol from the culture broth during fermentation. The fed-batch fermentation began as a batch operation and was switched to the fed-batch mode at 24 h, when OD600 reached 3.0. At the beginning of fed-batch fermentation, TB medium was supplemented with 10 g/L glycerol, 0.1% (v/v) trace element solution (27 g/L FeCl3·6H2O, 2 g/L ZnCl2·4H2O, 2 g/L CoCl2·6H2O, 2 g/L Na2MoO4·2H2O, 1 g/L CaCl2·2H2O, 1.3 g/L CuCl2·6H2O, and 0.5 g/L H3BO3) and 1% (v/v) vitamin solution (Sigma Aldrich, Cat. No. M6895). Glycerol was then fed at a rate of 0.01 L/h and 10% (v/v) canola oil (instead of 20% (v/v) n-dodecane) was used to overlay 30 L TB medium in a 50 L fermenter.
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study
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Extraction of (−)-α-bisabolol was performed using four common vegetable oils: canola, olive, corn, and soybean oil. A total of 0.1 g of (−)-α-bisabolol was added to 10 mL of sterile TB medium in a glass vial and 1 mL of individual vegetable oil was overlaid to the solution. After gentle stirring of the mixture, it was incubated at 25 °C for 1 h to separate the water-based TB medium and oil phase. The aqueous layer was removed and the organic layer was transferred to a new vial for gas chromatography (GC) analysis of (−)-α-bisabolol. The recovery yield of each vegetable oil was compared with that of n-dodecane. To determine the recovery efficiency of n-dodecane from cell pellets and TB medium, E. coli DH5α cells transformed with the pSNA-MrBBS-IspA plasmid were cultivated in TB medium in the presence of 20% (v/v) n-dodecane at 30 °C at 180 rpm for 48 h. After centrifugation of the culture at 14,000 rpm for 10 min, the pellet, supernatant, and organic layers of n-dodecane were fractionated. To determine the recovery yield of n-dodecane, the organic layer was directly subjected to GC analysis of (−)-α-bisabolol. The isolated supernatant was mixed with 20% (v/v) n-dodecane, incubated for 30 min, and centrifuged at 14,000 rpm for 10 min. The organic layer was isolated and used for GC analysis of (−)-α-bisabolol. The cell pellet was first disrupted by sonication and the lysate was used for the extraction and quantification of (−)-α-bisabolol through the process described above.
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To analyze (−)-α-bisabolol production, the culture broth was centrifuged at 13,000 rpm for 10 min to separate the overlaid n-dodecane phase. The recovered n-dodecane-phase was subsequently analyzed for (−)-α-bisabolol by GC and GC-mass spectrometry (GC–MS). The identification of (−)-α-bisabolol was conducted by GC–MS (5977A MSD) with a HP-5MS column (30 m × 0.250 mm × 0.25 µm, Agilent, Santa Clara, USA). The column flow was maintained at 1 mL/min. The oven temperature was initially held at 60 °C for 2 min, increased by 5 °C/min to 300 °C, and held at 300 °C for 10 min. (−)-α-Bisabolol (Cat. No. 23089-26-1, Sigma Aldrich) was used as the standard, and was confirmed through the GC-MS software, Mass Hunter (Agilent, Santa Clara, USA). The quantification of (−)-α-bisabolol was performed using GC equipped with a flame ionization detector (FID) with a HP-5 column (30 m × 0.320 mm × 0.25 µm) at a flow rate of 1 mL/min. The starting temperature of the oven was 60 °C for 2 min, increased at 5 °C/min to 200 °C, held at 200 °C for 2 min, increased by 50 °C/min to 300 °C, and held at 300 °C for 5 min. Helium was used as the carrier gas with an inlet pressure of 5.58 psi. The (−)-α-bisabolol concentration produced by engineered E. coli was determined as follows:\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$(-) {\text{-}} \alpha {\text{-bisabolol }}\left[{\text{mg}}/{\rm L} \right] = \frac{{\left[ {\left( - \right) {\text{-}} \alpha {\text{-bisabolol in }}n-{\text{ dodecane phase}}} \right] \times \left[ {{\text{volume of }}n-{\text{ dodecane phase}}} \right]}}{{\left[ {\text{volume of medium phase}} \right]}}$$\end{document}(-)-α-bisabololmg/L=--α-bisabolol inn-dodecane phase×volume ofn-dodecane phasevolume of medium phase
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| 100.0 |
The culture broth was centrifuged and the supernatant was used to quantify glycerol consumption and byproduct formation. Glycerol, acetate, and mevalonate concentrations were determined by high performance liquid chromatography (HPLC, Shimadzu, Kyoto, Japan) with a refractive index detector at 454 nm using Aminex HPX-87H column (1300 mm × 7.8 mm, Bio-Rad, Hercules, USA). Sulfuric acid (0.4 mM) was used as the mobile phase at a flow rate of 0.3 mL/min at 50 °C.
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study
| 99.94 |
(−)-α-Bisabolol is a naturally occurring sesquiterpene alcohol that exhibits antibacterial activity . This property would be a hurdle for the use of E. coli as a terpenoid production host . Therefore, we examined the suitability of three different E. coli strains [DH5α, MG1655, and BL21(DE3)] for (−)-α-bisabolol production by conducting growth assays in LB in the presence of various concentrations of (−)-α-bisabolol (Fig. 2). After 3 h of cultivation (mid-log phase), the growth of BL21(DE3) significantly decreased by 1 and 5 g/L (−)-α-bisabolol (Fig. 2a). The growth of MG1655 was not affected by (−)-α-bisabolol at concentrations of 1 or 5 g/L (Fig. 2a). The growth of DH5α slightly decreased at (−)-α-bisabolol concentration of 5 g/L, but growth was not affected by 1 g/L (−)-α-bisabolol (Fig. 2a). This result was corroborated with CFU measurements for each culture. As expected, DH5α showed the best tolerance to (−)-α-bisabolol whereas BL21(DE3) formed no colonies on the solid LB medium (Fig. 2b). These results are consistent with a previous study showing that of five different B- and K-type E. coli strains (MG1655, DH5α, S17-1, XL1-Blue, and BL21), DH5α is the best producer of the β-carotene (C40) terpenoid . In addition, E. coli DH5α has been used for production of retinol , farnesol , and protoilludene . We concluded that E. coli DH5α is suitable as a host for (−)-α-bisabolol production and it was used for all subsequent experiments.Fig. 2Growth inhibition of E. coli strains by exogenous (−)-α-bisabolol. a Time course of growth profile. b CFU measurement. Cells were grown in LB liquid medium and different amounts of (−)-α-bisabolol were added at the beginning of cultivation. Samples were taken for measurement of optical density at 600 nm (OD600). Samples for CFU measurement were taken at early stationary phase (indicated by an arrow). Mean values of two independent experiments are given for the CFU measurement
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| 100.0 |
Growth inhibition of E. coli strains by exogenous (−)-α-bisabolol. a Time course of growth profile. b CFU measurement. Cells were grown in LB liquid medium and different amounts of (−)-α-bisabolol were added at the beginning of cultivation. Samples were taken for measurement of optical density at 600 nm (OD600). Samples for CFU measurement were taken at early stationary phase (indicated by an arrow). Mean values of two independent experiments are given for the CFU measurement
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To implement the de novo production of (−)-α-bisabolol in E. coli DH5α, the gene of MrBBS, encoding the (−)-α-bisabolol synthase, was codon-optimized for E. coli and introduced via the pTSN-MrBBS plasmid (Fig. 1b). The resulting transformant cells were cultured in LB medium overlaid with 20% (v/v) n-dodecane for 48 h. Two-phase cultures have been successfully used to extract toxic, water-immiscible or volatile products [24–26] and n-dodecane was successfully used for extraction of (−)-α-bisabolol from the culture broth of S. cerevisiae . As expected, in the present study, (−)-α-bisabolol was extracted into the n-dodecane phase in the two-phase E. coli culture. As the (−)-α-bisabolol was recovered in n-dodecane without significant residual amounts left in the cells (Additional file 1: Figure S2), (−)-α-bisabolol purification in a later step will be facilitated. The n-dodecane phase was used for GC analysis to measure (−)-α-bisabolol concentrations. There was a peak at 25.6 min in the n-dodecane phase sample of E. coli DH5α with pTSN-MrBBS, corresponding to the standard (−)-α-bisabolol compound dissolved in n-dodecane (Fig. 3a). Mass spectrometry confirmed that the peak at 25.6 min was (−)-α-bisabolol (Fig. 3b, c) and a maximum concentration of 3 mg/L (−)-α-bisabolol was produced by the endogenous MEP pathway and exogenous MrBBS enzyme in E. coli DH5α. The peak was not observed in two-phase cultures of DH5α cells containing an empty plasmid as a control experiment (Fig. 3a). The (−)-α-bisabolol synthase gene, MrBBS, used in this study was recently isolated from German chamomile and, unlike other TSPs, exclusively synthesizes (−)-α-bisabolol as a single terpene product . Several TPSs producing α-bisabolol as a single major product have been previously cloned and biochemically characterized. However, these enzymes usually produce undesirable isomers with different structures or α-bisabolol of unknown stereochemistry [27–29]. The MrBBS enzyme has shown unique catalytic features including the formation of a single enantiopure (−)-α-bisabolol indicating the possibility of a biotechnological application to natural (−)-α-bisabolol production in S. cerevisiae . Here, we have expressed the codon-optimized synthetic MrBBS in E. coli and successfully produced (−)-α-bisabolol (titer: 3 mg/L). A similar result has been observed when attempting to express other plant terpene synthases in E. coli [30, 31].Fig. 3GC and GC–MS analysis of n-dodecane extracts from E. coli expressing the MrBBS gene. a Total ion chromatograms of an authentic (−)-α-bisabolol standard (100 mg/L) and the n-dodecane extracts from the E. coli expressing MrBBS (pTSN-MrBBS) or containing an empty vector (pTrc99A). b Mass spectra of (−)-α-bisabolol standard. c Mass spectra of (−)-α-bisabolol produced in E. coli cells harboring the plasmid pTSN-MrBBS. Each cell was cultivated in LB medium overlaid with 20% (v/v) n-dodecane at 30 °C for 48 h
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GC and GC–MS analysis of n-dodecane extracts from E. coli expressing the MrBBS gene. a Total ion chromatograms of an authentic (−)-α-bisabolol standard (100 mg/L) and the n-dodecane extracts from the E. coli expressing MrBBS (pTSN-MrBBS) or containing an empty vector (pTrc99A). b Mass spectra of (−)-α-bisabolol standard. c Mass spectra of (−)-α-bisabolol produced in E. coli cells harboring the plasmid pTSN-MrBBS. Each cell was cultivated in LB medium overlaid with 20% (v/v) n-dodecane at 30 °C for 48 h
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Terpenoid production in microbes is mainly limited by the flux from intermediates of central metabolism (acetyl-CoA for MVA pathway or pyruvate and glyceraldehyde-3-phosphate for MEP pathway) to the substrates of TPSs (GPP, FPP, or GGPP) . The introduction and engineering of the biosynthetic MVA pathway in E. coli have improved the production of terpenoids such as amorphadiene , carotenoids [19, 21], coenzyme Q10 , and farnesol by efficiently supplying IPP and DMAPP. Based on these studies, we exploited the biosynthetic MVA pathway for the biosynthesis of (−)-α-bisabolol in E. coli. To increase carbon flux towards TPS substrates, a plasmid (pSSN12Didi) containing the lower MVA pathway genes (Fig. 1b), leading from MVA to IPP and DMAPP , was introduced into E. coli DH5α expressing the MrBBS. MVA is an exogenous substrate for E. coli, so supplemented MVA can only be converted to IPP and DMAPP by the engineered MVA lower pathway in E. coli. Different concentrations of MVA, ranging from 0 to 10 mM, were examined to determine whether MVA availability limits (−)-α-bisabolol production in LB medium. (−)-α-bisabolol production increased as the concentration of the supplied MVA increased (Fig. 4a) and cell growth was not significantly inhibited (Fig. 4b). With the addition of 10 mM MVA, (−)-α-bisabolol production was 10.5 mg/L, approximately 3.5-fold higher than that obtained without the addition of MVA (3 mg/L, Fig. 4a). Expression of the lower MVA pathway genes in E. coli improved (−)-α-bisabolol production with the addition of MVA. However, MVA is not an economically viable substrate. To achieve (−)-α-bisabolol production from cost-effective renewable resources such as glycerol, a major byproduct of the biodiesel industry, we constructed a plasmid pSNA-MrBBS (Fig. 1b). pSNA-MrBBS harbors the complete biosynthetic MVA pathway genes, including the MrBBS gene, and enables production of IPP and DMAPP in the absence of MVA. A total of 12.8 mg/L of (−)-α-bisabolol was produced from glycerol in E. coli DH5α with pSNA-MrBBS grown in LB (Fig. 5a). This is a slightly higher concentration than that produced in DH5α containing only MrBBS and lower MVA pathway genes with the addition of 10 mM MVA (10.5 mg/L). Overall, the expression of biosynthetic MVA pathway genes significantly improved (−)-α-bisabolol production in E. coli.Fig. 4The effect of mevalonate concentration on (−)-α-bisabolol production (a) and final OD600 (b) from engineered E. coli harboring pTSN-MrBBS and pSSN12Didi. Cells were grown at 30 °C in LB medium supplemented with different concentrations of mevalonate and 20% (v/v) n-dodecane for 48 h. Data represent averages from three replicate cultures; error bars show SD Fig. 5Optimal conditions for (−)-α-bisabolol production in E. coli expressing MrBBS and all MVA pathway genes. E. coli cells harboring pSNA-MrBBS were grown for 48 h at 30 °C in the presence of 20% (v/v) n-dodecane with the following variations; a Medium (M9 supplemented with 4 g/L glucose, LB, 2xYT, TB) and glycerol concentration; b IPTG concentration. When OD600 reached 0.5, different concentrations of IPTG were added to the E. coli cells grown on TB medium supplemented with 10 g/L glycerol; c pH. Different initial pHs were tested to find an optimal pH for (−)-α-bisabolol production. E. coli cells were cultivated in TB medium containing 10 g/L glycerol in the absence of IPTG; d E. coli strains. E. coli cells were grown on TB medium, pH 7.0 with 10 g/L glycerol and 20% (v/v) n-dodecane in the absence of IPTG. Data represent averages from three replicate cultures and error bars show SD
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The effect of mevalonate concentration on (−)-α-bisabolol production (a) and final OD600 (b) from engineered E. coli harboring pTSN-MrBBS and pSSN12Didi. Cells were grown at 30 °C in LB medium supplemented with different concentrations of mevalonate and 20% (v/v) n-dodecane for 48 h. Data represent averages from three replicate cultures; error bars show SD
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| 100.0 |
Optimal conditions for (−)-α-bisabolol production in E. coli expressing MrBBS and all MVA pathway genes. E. coli cells harboring pSNA-MrBBS were grown for 48 h at 30 °C in the presence of 20% (v/v) n-dodecane with the following variations; a Medium (M9 supplemented with 4 g/L glucose, LB, 2xYT, TB) and glycerol concentration; b IPTG concentration. When OD600 reached 0.5, different concentrations of IPTG were added to the E. coli cells grown on TB medium supplemented with 10 g/L glycerol; c pH. Different initial pHs were tested to find an optimal pH for (−)-α-bisabolol production. E. coli cells were cultivated in TB medium containing 10 g/L glycerol in the absence of IPTG; d E. coli strains. E. coli cells were grown on TB medium, pH 7.0 with 10 g/L glycerol and 20% (v/v) n-dodecane in the absence of IPTG. Data represent averages from three replicate cultures and error bars show SD
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| 100.0 |
IPP and DMAPP, precursors of FPP, are essential metabolites in E. coli that are used for tRNA prenylation, synthesis of quinone and dolichol for respiration, and cell wall biosynthesis . Thus, the metabolic balance of IPP and DMAPP between endogenous essential metabolism and exogenous (−)-α-bisabolol production plays a crucial role in improving production of (−)-α-bisabolol and alleviation of growth inhibition. To determine the optimal IPP and DMAPP pool balancing conditions in the engineered E. coli harboring the entire MVA pathway and MrBBS genes (pSNA-MrBBS), we examined if cultivation media and glycerol availability limit (−)-α-bisabolol production. To do this we tested different media and concentrations of glycerol ranging from 0 to 3.5% (w/v) (Fig. 5a). E. coli DH5α containing the pSNA-MrBBS produced the highest concentration of (−)-α-bisabolol (24.2 mg/L) in TB medium without the addition of an additional carbon source (Fig. 5a). (−)-α-Bisabolol production was dependent on the amount of provided glycerol in the TB medium. With 1% (w/v) glycerol, (−)-α-bisabolol concentration was 38.3 mg/L, approximately 1.4-fold higher than the concentration of (−)-α-bisabolol obtained without the inclusion of glycerol. However, 3.5% (w/v) of glycerol concentration did not show further improvement of (−)-α-bisabolol production (Fig. 5a). Because cell growth of E. coli DH5α varied in different culture media, the differences in (−)-α-bisabolol production are partly due to differences in growth (Figure S3A).
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| 100.0 |
Plasmid pSNA-MrBBS contained IPTG-inducible lac and trc promoters for expression of the complete MVA pathway genes and MrBBS gene, respectively (Fig. 1b). To ascertain the optimal inducer concentration for (−)-α-bisabolol production in E. coli DH5α with pSNA-MrBBS, induction was conducted with varying concentrations of IPTG ranging from 0 to 0.1 mM. Interestingly, leaky expression of the MrBBS and MVA pathway genes in the absence of IPTG showed the highest production of (−)-α-bisabolol (38.9 mg/L). At all other IPTG concentrations (−)-α-bisabolol production decreased as the IPTG concentration increased (Fig. 5b) whereas the growth of E. coli DH5α cells is similar regardless of IPTG concentration (Additional file 1: Figure S3B). This result is compatible with a previous report that showed lycopene production in E. coli expressing lower MVA pathway genes regulated by the trc promoter decreased under all IPTG-induced conditions. In addition, constitutive promoters controlling the expression of MVA pathway genes showed much lower production of amorphadiene than that from IPTG-inducible lacUV5 and trc promoters . High expression of the MVA pathway and MrBBS genes under IPTG induced conditions could cause a deficiency of FPP for essential cellular metabolism. Furthermore, the accumulation of toxic intermediates of the MVA pathway, including IPP and 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) [32, 35], could cause growth inhibition along with a decrease of (−)-α-bisabolol production. In addition, we investigated the effects of initial pH of the cultivation of E. coli DH5α with pSNA-MrBBS on the production of (−)-α-bisabolol. The optimal pH for (−)-α-bisabolol production was pH 7 (38.2 mg/L, Fig. 5c). To determine the ideal E. coli strains for (−)-α-bisabolol production under the optimized culture conditions, we individually introduced the plasmid pSNA-MrBBS into three different E. coli strains (DH5α, MG1655, or BL21(DE3)) and measured the (−)-α-bisabolol concentration produced from each. E. coli DH5α cells produced the highest amount of (−)-α-bisabolol, and BL21(DE3) showed an approximate 13-fold decrease in (−)-α-bisabolol production compared to DH5α (38.4 mg/L, Fig. 5d). We also measured cell growth during examination of pH effects on (−)-α-bisabolol production in E. coli DH5α expressing MrBBS and entire MVA pathway genes. Although cell growth slightly decreased at a pH 5 or 6, it was similar at all other pHs (Additional file 1: Figure S3C).
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(−)-α-Bisabolol is produced from depyrophosphorylation of the prenyl diphosphate precursor FPP. Therefore, synthesis of FPP from IPP and DMAPP by FPP synthase, encoded by the ispA gene, plays a key role in the improved production of (−)-α-bisabolol. Furthermore, high concentrations of prenyl diphosphates, such as FPP, are toxic to E. coli cell growth . FPPs needs to be efficiently converted into downstream products by TPSs to reduce its toxicity. To increase the synthesis of the cellular terpenoid biosynthesis intermediate FPP, and (−)-α-bisabolol production, overexpression of an FPP synthase, encoded by the ispA gene, was performed. The ispA gene of E. coli was inserted downstream of the MrBBS gene in the plasmid pSNA-MrBBS, resulting in the pSNA-MrBBS-IspA plasmid (Fig. 1b). This plasmid was transformed into E. coli DH5α and cultivated in TB medium containing 1% (w/v) glycerol at 30 °C for 48 h using a two-phase culture without the addition of IPTG. Overexpression of the ispA gene improved (−)-α-bisabolol production compared with pSNA-MrBBS alone. After 48 h, a concentration of 79.7 mg/L of (−)-α-bisabolol was obtained in a culture of DH5α with pSNA-MrBBS-IspA. This was twofold higher than the 39.7 mg/L of (−)-α-bisabolol produced by DH5α with pSNA-MrBBS alone (Fig. 6a), without significant difference in cell growth (Additional file 1: Figure S3D). This suggested that sufficient supply of FPP is important to improve (−)-α-bisabolol production in E. coli. While overexpressing the ispA gene, 0.05 mM IPTG at OD600 ~0.5 was added to induce expression of the MVA pathway and MrBBS genes. Similar to the results without ispA overexpression, (−)-α-bisabolol production decreased from 87.8 mg/L (no IPTG) to 40 mg/L (−)-α-bisabolol (0.05 mM IPTG, Fig. 6b) with no significant difference in cell growth (Additional file 1: Figure S3D). To monitor the time-course of (−)-α-bisabolol production, batch fermentation of E. coli DH5α harboring the pSNA-MrBBS-IspA plasmid, using a 1 L fermenter, was conducted at 30 °C in the absence of IPTG for 93 h (Fig. 6c). (−)-α-bisabolol production of 214 mg/L was achieved and cell growth reached an OD600 value of 11.5 after 93 h. MVA and acetate were the main byproducts, although the acetate was consumed after glycerol depletion at 48 h. The solubility of heterologous enzymes increases at lower growth temperatures because the transcription rate is slower, leading to better protein folding in E. coli [37, 38]. In addition, improved solubility of metabolic pathway enzymes at low temperature enhances the production yield . Therefore, we performed the batch fermentation at 25 °C under the same conditions. Indeed, (−)-α-bisabolol production was 342 mg/L after fermentation for 93 h, approximately 1.6-fold higher than that obtained at 30 °C, and cell growth decreased by 25% (OD600 8.7, Fig. 6d).Fig. 6IspA overexpression and batch fermentation of engineered E. coli. a Improvement of (−)-α-bisabolol production in E. coli DH5α transformed with pSNA-MrBBS-IspA compared with DH5α harboring the pSNA-MrBBS plasmid. Cells were cultivated in TB medium containing 10 g/L glycerol at 30 °C for 48 h using two-phase culture without the addition of IPTG. Data represent averages from three replicate cultures; error bars show SD, b effects of IPTG addition on (−)-α-bisabolol production in E. coli DH5α harboring pSNA-MrBBS-IspA. Culture conditions were the same as in Fig. 6a, except for the addition of IPTG. IPTG was added to each culture to a final concentration of 0.05 mM when the OD600 reached 0.5. Data represent averages from three replicate cultures; error bars show SD, c time profile of batch fermentation of E. coli DH5α with pSNA-MrBBS-IspA in TB medium containing 10 g/L of glycerol as carbon source at 30 °C, n-dodecane was overlaid up to 20% (v/v) of culture volume to continuously extract (−)-α-bisabolol from the culture broth during fermentation. d Time profile of batch fermentation of E. coli DH5α with pSNA-MrBBS-IspA in TB medium containing 10 g/L of glycerol as carbon source at 25 °C. n-Dodecane was overlaid up to 20% (v/v) of culture volume to continuously extract (−)-α-bisabolol from the culture broth during fermentation
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IspA overexpression and batch fermentation of engineered E. coli. a Improvement of (−)-α-bisabolol production in E. coli DH5α transformed with pSNA-MrBBS-IspA compared with DH5α harboring the pSNA-MrBBS plasmid. Cells were cultivated in TB medium containing 10 g/L glycerol at 30 °C for 48 h using two-phase culture without the addition of IPTG. Data represent averages from three replicate cultures; error bars show SD, b effects of IPTG addition on (−)-α-bisabolol production in E. coli DH5α harboring pSNA-MrBBS-IspA. Culture conditions were the same as in Fig. 6a, except for the addition of IPTG. IPTG was added to each culture to a final concentration of 0.05 mM when the OD600 reached 0.5. Data represent averages from three replicate cultures; error bars show SD, c time profile of batch fermentation of E. coli DH5α with pSNA-MrBBS-IspA in TB medium containing 10 g/L of glycerol as carbon source at 30 °C, n-dodecane was overlaid up to 20% (v/v) of culture volume to continuously extract (−)-α-bisabolol from the culture broth during fermentation. d Time profile of batch fermentation of E. coli DH5α with pSNA-MrBBS-IspA in TB medium containing 10 g/L of glycerol as carbon source at 25 °C. n-Dodecane was overlaid up to 20% (v/v) of culture volume to continuously extract (−)-α-bisabolol from the culture broth during fermentation
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n-Dodecane has been mostly used for the extraction of terpenoids produced in microorganisms due to its relatively low volatility, enabling continuous extraction over multiple days . The (−)-α-bisabolol produced in flask culture and batch fermentation could be efficiently extracted from the broth to the overlaid n-dodecane phase. The in situ recovery of (−)-α-bisabolol from the n-dodecane phase during culture can reduce production costs by simplifying both the harvest and purification processes. However, n-dodecane is not an economically viable extracting solvent. We assessed various natural and inexpensive vegetable oils for selective (−)-α-bisabolol extraction from fermentation broth. As shown in Table 2, all tested vegetable oils showed extraction yields ranging from 96.6 to 98.8%, which are comparable to that of n-dodecane (99.5%). Among them, canola oil (98.8%) was the best extracting oil and was used for the extraction of (−)-α-bisabolol during fed-batch fermentation. A greener extraction of (−)-α-bisabolol plays a key role in the application of (−)-α-bisabolol produced from fermentation broth to pharmaceutical and cosmetic products. We successfully used vegetable oils as natural, cost-effective, and biodegradable extractors for in situ extraction of (−)-α-bisabolol during fermentation. Moreover, due to the difference in polarity between the fermentation broth (water based) and vegetable oils, there is a phase separation, resulting in isolation of oil from the broth containing cell debris. This in situ (−)-α-bisabolol separation from the fermentation broth can also alleviate production inhibition and improve (−)-α-bisabolol production, which will be of interest in industrial processes. To examine the production performance of (−)-α-bisabolol under conditions more relevant to industrial processes, we conducted (−)-α-bisabolol fermentation in a 50 L fermenter with a fed-batch mode. E. coli DH5α, containing pSNA-MrBBS-IspA, was grown at 25 °C with 10% (v/v) canola oil as an extracting solution. The final (−)-α-bisabolol titer reached 3.3 g/L after 90 h of fermentation, which was almost 8.4-fold higher than that obtained from the batch fermentation at 25 °C (Fig. 7). At the beginning of fermentation, MVA was accumulated until depletion of the initial 10 g/L glycerol. After complete depletion of initial glycerol, MVA was consumed and (−)-α-bisabolol accumulated continuously up to 9.1 g/L at the end of fermentation (Fig. 7). The specific (−)-α-bisabolol production and productivity reached 0.18 g/g dry cell weight (DCW) and 1.24 g/L/day, respectively. A previous study reported the production of 8 mg/L of (−)-α-bisabolol from S. cerevisiae by overexpressing the MrBBS gene for 4 days . In this study, two-phase fed-batch fermentation of the DH5α strain expressing the MrBBS, entire MVA pathway, and ispA genes yielded 9.1 g/L of (−)-α-bisabolol after 150 h, a 1137-fold increase from that reported in the previous study. Since the (−)-α-bisabolol intermediate, mevalonate, accumulated after glycerol depletion, (−)-α-bisabolol production would increase if we increase expression of the genes of the lower MVA pathway. Such adjustments will be the content of further work to improve (−)-α-bisabolol production.Table 2Recovery yield of (−)-α-bisabolol using common vegetable oilsExtracting solutionExtraction yield (%)Soybean oil98.5Canola oil98.8Corn oil98.5Sunflower oil96.7 n-Dodecane99.5 Fig. 7Fed-batch fermentation of E. coli DH5α in TB medium supplemented with 10 g/L of glycerol as initial carbon source. After complete depletion of glycerol, glycerol was fed at 3 g/L/h. Concentrations of acetate, mevalonate, and glycerol were determined by HPLC and (−)-α-bisabolol content was measured using GC. Canola oil 10% (v/v) instead of n-dodecane 20% (v/v) was used to overlay 30 L TB medium in a 50 L fermenter
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Fed-batch fermentation of E. coli DH5α in TB medium supplemented with 10 g/L of glycerol as initial carbon source. After complete depletion of glycerol, glycerol was fed at 3 g/L/h. Concentrations of acetate, mevalonate, and glycerol were determined by HPLC and (−)-α-bisabolol content was measured using GC. Canola oil 10% (v/v) instead of n-dodecane 20% (v/v) was used to overlay 30 L TB medium in a 50 L fermenter
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study
| 99.94 |
In this study, we have engineered E. coli for de novo production of (−)-α-bisabolol for the first time. Introduction of the MVA pathway, (−)-α-bisabolol synthase from German chamomile, and FPP synthase turned E. coli into a microbial cell factory for the de novo production of (−)-α-bisabolol from the renewable carbon source, glycerol. Using Canola oil as an extraction solvent, the engineered E. coli strain produced 9.1 g/L of (−)-α-bisabolol from glycerol in a fed-batch fermentation system, and the specific (−)-α-bisabolol production and productivity reached 0.18 g/g DCW and 1.24 g/L/day, respectively. To the best of our knowledge, this is the first report demonstrating that a large amount of (−)-α-bisabolol could be produced by metabolically engineered E. coli. Although more work is needed to optimize the fermentation process, the strains developed in this study will serve as promising platform hosts for development of microbial production of (−)-α-bisabolol on a large scale. Moreover, a more environmentally friendly means to extract (−)-α-bisabolol, using vegetable oils, will reduce the production cost in an industrial process.
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| 100.0 |
Electronic cigarette (E-cigarette) is a term that usually refers to a kind of electronic product that imitates traditional cigarettes in terms of their appearance, taste, aroma and other aspects. They are powered by batteries, conveying nicotine to the respiratory system via a heating or atomizing method, which leads to the same physiological and psychological feeling as traditional cigarettes . In 2014, the World Health Organization (WHO) defined devices that could release nicotine (also known as E-cigarettes) as products that suck nicotine aerosol through a cigarette holder or other components .
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other
| 99.9 |
Traditional tobacco products mainly release chemicals for people to smoke via chemical reaction triggered by burning; while E-cigarettes output nicotine and aromatic substances by means of electronic heating, and are electronic devices that enable smokers to have the same physiological feeling as smoking real cigarettes. They were first invented by Chinese pharmacist Han Li, and have gradually flowed into Europe and America. Although the exact structural design of E-cigarettes is constantly changing, the structure is composed of three parts: typically, a battery, atomizer, and liquid storage tank (cartridge) (as shown in Figure 1) . When people smoke E-cigarettes, the electronic device in the E-cigarette smoke will atomize the e-liquid (the solution used to generate the aerosol) in the cartridge, and then the smoke will enter the body through the mouth. In recent years, many enterprises have entered the field of E-cigarettes. In order to cater to the diverse needs of people, E-cigarettes have been developed with a variety of tastes. The production technology of E-cigarettes has been constantly improved, and its market share continues to expand.
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review
| 99.5 |
The e-liquid of the E-cigarette is usually a liquid mixture of propylene glycol, glycerin, nicotine, essence and some other chemical substances. E-cigarette sellers have always packaged E-cigarettes as harmless smoking-cessation products or cigarette substitutes without harmful substances such as tar, carbon monoxide, suspended particles, etc. However, up to now, the research about the safety of E-cigarettes has been scarce, and there is no systematic review focusing on the safety assessment of E-cigarettes worldwide . In particular, it is still unclear whether aerosols produced by electronic smoke do harm to our health. Furthermore, it’s controversial that E-cigarettes emit only harmless steam . Although more and more research shows that the known dangers of electronic smoke are much smaller than for traditional cigarettes, it is uncertain how dangerous the potential toxic effects of E-cigarettes are.
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review
| 99.9 |
Nicotine is highly involved in the development of cardiovascular disease, especially atherosclerosis. Although tar and other harmful ingredients have been removed in the e-liquid, more work should be done to evaluate its safety, due to the existence of nicotine and other potential hazards such as aerosol particles and different flavor additives in the e-liquid. These may increase the risk of cardiovascular disease. Moreover, the number and size distribution of particles from E-cigarettes are similar to those from traditional cigarettes, and some E-cigarettes produce more particles than traditional cigarettes. Particles may cause lung and systemic inflammation, and increase the risk of cardiovascular disease, respiratory disease and death .
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review
| 99.25 |
As a result, it is of great importance to evaluate whether E-cigarettes can become a substitute for traditional cigarettes and become a safe and reliable way to quit smoking. Therefore, the aim of current study is to systematically review the literature reports about the safety of electronic cigarettes and to understand its hazards and disadvantages.
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review
| 99.9 |
With the extension of people’s health consciousness and the worldwide campaigns for a blanket smoking ban, E-cigarettes first appeared in the public arena. The basic concept of E-cigarettes is that they are smokeless, non-tobacco cigarettes, and this was proposed by Herbert Gilbert in 1963, and patented in 1965, but did not enter the market . In 2003, a Chinese pharmacist Han Li applied for and obtained a patent for “a non-flammable electronic atomizing cigarette” . In 2004, the Ruyan Company of China began to develop and sell E-cigarettes. E-cigarettes gradually became commercially available. In 2007, the international patent of E-cigarettes was obtained . Since then, the E-cigarettes have continued to develop, spreading from China to Europe, America, Japan and other countries. By 2014, E-cigarettes were being sold in more than 60 countries and regions all over the world, and were being sought after by more and more consumers of different ages. China has become the main production place of E-cigarettes, and about 90% of E-cigarettes in the world are produced in Shenzhen . In recent years, in response to the different demands of E-cigarette consumers, along with the broad market requirements, E-cigarettes have developed rapidly. Different electronic products have been put on the market, and the replacement cycle of similar products is very short.
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review
| 99.0 |
The regulation strategy for E-cigarettes is not unified all over the world. Except for some countries that either completely prohibit E-cigarettes or have not yet declared, the other countries variously regard it as tobacco, a pharmaceutical product, or as an ordinary consumer product (see Table 1) . The following part will introduce the E-cigarette regulation strategy in different countries. Some countries adopt a “classification regulation” strategy, classifying the E-cigarettes according to the existence of nicotine or tobacco extract in e-liquid or, the content and concentration of nicotine. Different types of E-cigarettes comply with different control policies. The classification standards of E-cigarettes in different countries are quite different. Generally, E-cigarettes can be classified into one of three categories: tobacco, pharmaceutical products, or ordinary consumer goods.
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other
| 96.3 |
The United States, South Korea and Singapore regard E-cigarettes as tobacco under control (see Table 1). The European Union implements classified control on E-cigarettes. The Tobacco Products Directive (TPD) stipulates that E-cigarettes containing nicotine will be included in the control range from May 2016. There are also differences between countries. In Korea, Togo and other countries, E-cigarettes containing nicotine are regarded as tobacco, while in Lithuania, Malta, Thailand, Vietnam and other countries, E-cigarettes are generally regarded as tobacco.
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other
| 99.9 |
Japan and other countries regard E-cigarettes as pharmaceutical products (see in Table 1). Among them, some countries and regions regard E-cigarettes as medical products, such as Denmark, Estonia, Greece, Hungary, New Zealand, the Philippines, Slovakia, Sweden, and Taiwan, China. Other countries, such as Austria, Canada, Finland, Japan, and Switzerland, consider E-cigarettes to be pharmaceutical products based on whether the e-liquid contains nicotine or tobacco extracts. They regard E-cigarettes containing nicotine to be pharmaceutical products. For example, in France, when the E-cigarette and e-liquid are used for the purpose of quitting smoking, no matter how high the content and concentration of nicotine is, they will be regarded as pharmaceutical products .
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other
| 99.75 |
Italy, Russia, Spain and other countries regard E-cigarettes as electronic products. Switzerland regards E-cigarettes containing no nicotine as ordinary consumer goods. In France, when the E-cigarette and e-liquid are not regarded as pharmaceutical products, E-cigarettes are regarded as ordinary consumer goods, and are regulated by the general consumer product safety act.
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other
| 99.94 |
Under certain conditions, E-cigarettes may have attributes other than those of tobacco products, pharmaceutical products or ordinary consumer goods. For example, in Australia, Canada, and Malaysia, some E-cigarettes are regarded as being drugs, because the e-liquid contains a certain amount of nicotine.
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other
| 99.94 |
The output of E-cigarettes is huge. The policies and intensity of control are quite different due to the variation in their classification among different countries. At present, there are four main types of policy for the control of E-cigarettes. The first one is prohibition, which means prohibition of the import, sale, usage, advertisement, and public consumption, as well as the sale of E-cigarettes to minors. The second type of policy is to control E-cigarettes as drugs or medical products. The third one is to control E-cigarettes as tobacco products. The final category of policy is to regulate E-cigarettes as electronic products and ordinary consumer goods . The distribution of different E-cigarette control policies in the world are shown in Figure 2 .
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other
| 99.9 |
There are very few countries and regions that regulate the production of E-cigarettes. Brazil, Greece, Israel, the United Arab Emirates, and some local governments in India have banned E-cigarette production. Chile and Malaysia have also introduced management regulations on E-cigarette production. The control of E-cigarettes in China does not exist, and the product quality has no standards, meaning that the E-cigarette industry is in chaos. The quality of E-cigarette products is uneven and without security guarantees, and the hidden dangers behind this situation are worrying . In a report on the adverse effects of E-cigarette smoke issued by the United States Food and Drug Administration (FDA), it was pointed out that about half of the tobacco-related adverse events reported in more than 100 tobacco reports were related to E-cigarettes .
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other
| 99.8 |
In addition, the explosion of electronic smoke has been reported. In 16 January 2017, a man in Pocatello, Idaho State of the United States, lost 7 teeth and got second-degree burns on the left side of his left face due to the sudden explosion of an E-cigarette, and the great power even blew up the wash basin in the bathroom . Thus, although E-cigarettes have only been on the market for a short time, there are many quality and safety problems.
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other
| 99.9 |
The definite components in the e-liquid include propylene glycol, glycerol, nicotine (either included or not), and essence. Han, S.L. et al. determined the contents of the main components in 51 kinds of E-cigarette e-liquid. It was found that 1,2-propylene glycol and glycerol were the main solvents in most of E-cigarette e-liquids, with the content ranging between 75% and 95% . Although 1,2-propylene glycol and glycerol are widely used in the food, pharmaceutical and cosmetic industries, they are also food additives approved by the European Union . Although they are considered safe for oral use, propylene glycol will be converted into propylene oxide during aerosol inhalation, and propylene oxide is classified as a Class 2B carcinogen by the International Agency for Cancer Research. In addition, the glycerol will be converted to acrolein, which causes stimulation of the upper respiratory tract . A short-term exposure study showed that 5 min of E-cigarette use resulted in a significant increase in airway resistance . Another study found that with exposure to E-cigarette smoke, the number of free radicals in the lungs would increase significantly, indicating that E-cigarettes caused the development of oxidative stress in the lung. The content of 8-hydroxy-2-deoxyguanosine in the lungs also increased significantly. The 8-hydroxy-2-deoxyguanosine is widely used as a biomarker to assess oxidative stress and carcinogenicity . Goniewicz, M.L. et al. found four kinds of toxic substances, including carbonyl compounds, volatile organic compounds (VOCs), tobacco-specific nitrosamines (TSNAS), and heavy metals in the analysis of E-cigarette smoke . In addition, the essence, nicotine, and aerosol particles generated by electronic devices are harmful to the health .(1)For the carbonyl compounds, the heating of glycerol will produce harmful aldehydes related to temperature, including formaldehyde, acetaldehyde and acrolein. These three compounds were identified in almost all of the examined E-cigarettes . The study of Gassee, F.R. et al. showed that the mixture of the above three compounds produced more sensory stimuli than those of a single compound . Among them, formaldehyde was categorized as a Class 1 carcinogen in 2006. Acrolein stimulates the nasal cavity, damages the lungs and the inner walls of the blood vessels, and is a major factor leading to cardiovascular disease. Chronic inhalation of acrolein inhibits circulation of endothelial progenitor cells and promotes atherosclerosis, which accelerates the rate of hardening of the aorta by 1.6 times . Ambrus, J.L. et al. also confirmed the presence of acrolein, toxic substances and mutagenic compounds in E-cigarette smoke. It was found that the carcinogenic metabolic enzymes in rats exposed to E-cigarette smoke increased significantly, inducing a greater carcinogenic risk of carcinogens in E-cigarette smoke .(2)Nicotine is the addictive component of tobacco. It induces adverse effects during pregnancy and contributes to the progress of cardiovascular disease. Although nicotine itself is not a carcinogen, it functions as a “tumor promoter”. Nicotine is involved in neurodegeneration and other malignant diseases. Therefore, the use of nicotine in children, adolescents, pregnant women and women of reproductive age should be treated with great caution, due to the effects of nicotine on the brain . The effects of nicotine on the cardiovascular system will be elaborated in the following chapters.(3)Volatile organic compounds (VOCs) include toluene and meta xylene. In the study of Czogala, J. et al. almost all of the detected smokes contained toluene . In the study of Goniewicz, M.L. et al. toluene and meta xylene were detected in almost all sample smokes. These VOCs are irritating to the skin and mucous membrane, and have an anesthetic effects on the central nervous system, as well as having certain carcinogenicity .(4)N′-nitrosonornicotine (NNN), 4-(methylonitrosoamino)-1-(3-pirydyl)-l-butanone (NNK) and N′-nitrosoanatabine (NAT) are the three most representative tobacco-specific nitrosamines (TSNAS) in E-cigarettes. Kim, H.J. et al. tested the content of TSNAS in e-liquid samples. There were various kinds of TSNAS tested in 105 samples, among which the maximum concentration of NAT reached 62.19 ug/L, significantly exceeding the requirement for cigarette companies . Nitrosamines have strong carcinogenicity. Moreover, both nitrosamine and the polycyclic aromatic hydrocarbon benzopyrene can interact with DNA. These produced additives interfere DNA replication and the duplicated DNA produces purine free sites and triggers gene mutations . The chemical mixture in the smoke of the E-cigarette will cause chromosome division, and may cause damage to mitotic spindles or filaments, thereby inducing mutations .(5)Heavy metals include lead, nickel, cadmium, and so on. In 2013, Williams, M. et al. found heavy metals such as tin, nickel, lead, chromium and other nanoparticles in the smoke of E-cigarettes . The inhaled heavy metal nanoparticles can be deposited in the alveoli, inducing lung damage and leading to cough, dyspnea, chest pain, pulmonary edema, acute respiratory failure, as well as carcinogenicity, nephrotoxicity, and neurotoxicity . For example, tin is cytotoxic to human lung fibroblasts . In 2017, Williams, M. et al. continued to study the electronic flue gas sol. They selected 36 kinds of elements to detect, of which 35 kinds were detected in the electronic flue gas sol, while there were only 15 kinds of heavy metals detected in traditional cigarettes. These elements contain a variety of heavy metals, and the concentration is usually higher than that of traditional cigarette smoke . The content of lead and chromium is equal to that of traditional cigarette smoke. Especially the nickel content is much higher than that of traditional cigarettes . However, we still do not know the effects of inhaling heavy metal particles in aerosol on health.(6)There are studies that have investigated the cytotoxicity of the essence in E-cigarettes. Bahl, V. et al. used MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide) assays to test the cytotoxicity of e-liquids. The results showed that some e-liquids were cytotoxic to human embryonic stem cells and mouse neural stem cells, and that the composition and concentration of chemical components played an important role. In addition, more evidences confirmed that the chemical properties and the concentration of essence added to the e-liquid, rather than the nicotine alone, are much more harmful . Farsalinos, K.E. et al. used MTT tests to study the in vitro toxicity of the e-liquid of 20 E-cigarettes. The results showed that some electronic smoke liquid had a toxic effect on cardiac muscle cells after heating and atomizing, which would cause disease. The evidence indicated that the ingredients involved in the progress of diseases were related to the production process adopted by the liquid tobacco production company, as well as the flavor components added to cater to the public’s preference . Cinnamylaldehyde and diacetyl are approved flavorings in food, but they will affect people’s health when inhaled . Zeng, W.L. et al. collected tobacco smoke by cell culture medium, and studied the effect of smoke on the relative proliferation rate of Chinese hamster ovary cells (CHO cells) by MTT. The results showed that the relative proliferation rate of flue gas trapping liquid on CHO cells was 10 times higher than that of a 3R4F-reference-cigarette at 100% smoke concentration .
|
review
| 99.25 |
For the carbonyl compounds, the heating of glycerol will produce harmful aldehydes related to temperature, including formaldehyde, acetaldehyde and acrolein. These three compounds were identified in almost all of the examined E-cigarettes . The study of Gassee, F.R. et al. showed that the mixture of the above three compounds produced more sensory stimuli than those of a single compound . Among them, formaldehyde was categorized as a Class 1 carcinogen in 2006. Acrolein stimulates the nasal cavity, damages the lungs and the inner walls of the blood vessels, and is a major factor leading to cardiovascular disease. Chronic inhalation of acrolein inhibits circulation of endothelial progenitor cells and promotes atherosclerosis, which accelerates the rate of hardening of the aorta by 1.6 times . Ambrus, J.L. et al. also confirmed the presence of acrolein, toxic substances and mutagenic compounds in E-cigarette smoke. It was found that the carcinogenic metabolic enzymes in rats exposed to E-cigarette smoke increased significantly, inducing a greater carcinogenic risk of carcinogens in E-cigarette smoke .
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study
| 99.94 |
Nicotine is the addictive component of tobacco. It induces adverse effects during pregnancy and contributes to the progress of cardiovascular disease. Although nicotine itself is not a carcinogen, it functions as a “tumor promoter”. Nicotine is involved in neurodegeneration and other malignant diseases. Therefore, the use of nicotine in children, adolescents, pregnant women and women of reproductive age should be treated with great caution, due to the effects of nicotine on the brain . The effects of nicotine on the cardiovascular system will be elaborated in the following chapters.
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review
| 99.75 |
Volatile organic compounds (VOCs) include toluene and meta xylene. In the study of Czogala, J. et al. almost all of the detected smokes contained toluene . In the study of Goniewicz, M.L. et al. toluene and meta xylene were detected in almost all sample smokes. These VOCs are irritating to the skin and mucous membrane, and have an anesthetic effects on the central nervous system, as well as having certain carcinogenicity .
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study
| 99.94 |
N′-nitrosonornicotine (NNN), 4-(methylonitrosoamino)-1-(3-pirydyl)-l-butanone (NNK) and N′-nitrosoanatabine (NAT) are the three most representative tobacco-specific nitrosamines (TSNAS) in E-cigarettes. Kim, H.J. et al. tested the content of TSNAS in e-liquid samples. There were various kinds of TSNAS tested in 105 samples, among which the maximum concentration of NAT reached 62.19 ug/L, significantly exceeding the requirement for cigarette companies . Nitrosamines have strong carcinogenicity. Moreover, both nitrosamine and the polycyclic aromatic hydrocarbon benzopyrene can interact with DNA. These produced additives interfere DNA replication and the duplicated DNA produces purine free sites and triggers gene mutations . The chemical mixture in the smoke of the E-cigarette will cause chromosome division, and may cause damage to mitotic spindles or filaments, thereby inducing mutations .
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study
| 99.94 |
Heavy metals include lead, nickel, cadmium, and so on. In 2013, Williams, M. et al. found heavy metals such as tin, nickel, lead, chromium and other nanoparticles in the smoke of E-cigarettes . The inhaled heavy metal nanoparticles can be deposited in the alveoli, inducing lung damage and leading to cough, dyspnea, chest pain, pulmonary edema, acute respiratory failure, as well as carcinogenicity, nephrotoxicity, and neurotoxicity . For example, tin is cytotoxic to human lung fibroblasts . In 2017, Williams, M. et al. continued to study the electronic flue gas sol. They selected 36 kinds of elements to detect, of which 35 kinds were detected in the electronic flue gas sol, while there were only 15 kinds of heavy metals detected in traditional cigarettes. These elements contain a variety of heavy metals, and the concentration is usually higher than that of traditional cigarette smoke . The content of lead and chromium is equal to that of traditional cigarette smoke. Especially the nickel content is much higher than that of traditional cigarettes . However, we still do not know the effects of inhaling heavy metal particles in aerosol on health.
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study
| 99.56 |
There are studies that have investigated the cytotoxicity of the essence in E-cigarettes. Bahl, V. et al. used MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide) assays to test the cytotoxicity of e-liquids. The results showed that some e-liquids were cytotoxic to human embryonic stem cells and mouse neural stem cells, and that the composition and concentration of chemical components played an important role. In addition, more evidences confirmed that the chemical properties and the concentration of essence added to the e-liquid, rather than the nicotine alone, are much more harmful . Farsalinos, K.E. et al. used MTT tests to study the in vitro toxicity of the e-liquid of 20 E-cigarettes. The results showed that some electronic smoke liquid had a toxic effect on cardiac muscle cells after heating and atomizing, which would cause disease. The evidence indicated that the ingredients involved in the progress of diseases were related to the production process adopted by the liquid tobacco production company, as well as the flavor components added to cater to the public’s preference . Cinnamylaldehyde and diacetyl are approved flavorings in food, but they will affect people’s health when inhaled . Zeng, W.L. et al. collected tobacco smoke by cell culture medium, and studied the effect of smoke on the relative proliferation rate of Chinese hamster ovary cells (CHO cells) by MTT. The results showed that the relative proliferation rate of flue gas trapping liquid on CHO cells was 10 times higher than that of a 3R4F-reference-cigarette at 100% smoke concentration .
|
review
| 98.1 |
Secondhand smoke produced by traditional cigarettes has many effects on human health, including increased risk of respiratory tract diseases, lung cancer, infectious diseases, acute cardiovascular diseases, and cerebral apoplexy . The secondhand smoke of traditional cigarettes is mainly the side-stream smoke of the cigarette; however, the E-cigarette does not produce side-stream smoke. As a result, the second-hand smoke of the E-cigarette is composed of the exhaled fog of the E-cigarette user. We have evaluated the safety of secondhand smoke produced by E-cigarettes. Schripp, T. et al. placed subjects in a closed chamber to smoke E-cigarettes and exposed them to second-hand smoke. It has been shown that aerosol particles in the electronic smoke enter the lung tissue from the respiratory tract, cycle in the lungs and become smaller particles after being exhaled. In indoor environments, people can also “inhale the steam passively”. The analysis of air components showed that formaldehyde, acrolein, isoprene, acetaldehyde and acetic acid were present, but the chemical compounds produced by traditional cigarette burning were 5 to 40 times than those of the electronic ones . The study results of Goniewicz, M.L. et al. found that the release of smoke from E-cigarettes can increase the amount of nicotine deposition on windows, walls, floors, wood and metal surfaces. Therefore, they believe that smoking E-cigarettes may expose humans to the risk of nicotine indirectly, and future studies should explore the potential risks of carcinogens formed by the nicotine that is released from E-cigarettes . Schober, W. et al. conducted indoor research 6 times. It was found that after 2 h suction of E-cigarettes, the content of PM2.5 (particles less than 2.5 microns), propylene glycol, glycerol and nicotine in the indoor air increased significantly. In addition, the total polycyclic aromatic hydrocarbon (PAHs) content, which may cause cancer, increased by 20%, and the concentration of aluminum in the environment increased by 2.4 times . The results of the 5 experiments conducted by Czogala, J. et al. showed that after the use of E-cigarettes, the level of nicotine in the environment was 10% (3.3 compared to 31.6 g/m3) that of traditional cigarettes, and the PM2.5 concentration was about 18% of that of traditional cigarettes . In the study of Flouris, A.D. et al. and colleagues, 15 non-smokers were exposed to secondhand smoke from traditional cigarette or smoke from E-cigarettes for an hour, and then the cotinine levels in their serum were tested. The results showed that the cotinine levels in the serum of non-smokers were similar after inhaling the secondhand smoke of traditional cigarettes and E-cigarettes (2.6 to 2.4 ng/mL) . As a result, nicotine levels, along with levels of some potentially toxic substances in the body, will increase when they are exposed to the secondhand smoke of E-cigarettes. Although current studies have shown that the risk of secondhand smoke generated by E-cigarettes is expected to be lower than that of traditional cigarettes, there is a high possibility that non-smokers will be exposed to certain doses of nicotine and other toxic substances. With more and more E-cigarettes used in public spaces, a greater diffusion of these toxic and harmful substances will be released into the environment.
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study
| 99.9 |
Smoking cessation is the most effective way to prevent cardiovascular disease, so it is strongly recommended by guidelines for the prevention and treatment of cardiovascular disease . However, nicotine addiction makes it difficult for most people to quit smoking . Therefore, the current methodology of quitting smoking is a combination of nicotine replacement with the drugs that include nicotinic receptor agonists and behavioral therapy. In conjunction with this treatment, electronic smoke inhalation of nicotine simulates the effect of traditional cigarette smoking. As a result, more and more consumers prefer E-cigarettes when they quit smoking. It is undeniable that E-cigarettes rather than traditional tobacco are much more effective for some people to achieve smoking cessation. However, some people will become a “double user”, namely they smoke both traditional cigarettes and E-cigarettes, especially in places where smoking traditional cigarettes is prohibited. This does not help to quit smoking, but rather leads to greater harm from smoking. Because of the “harmless” nature of E-cigarettes, tobacco users may reuse tobacco products. E-cigarettes may also be a potential way for adolescents or non-smokers to use tobacco, and the use of E-cigarettes may also be the gateway to other drugs or harmful substances. Moreover, users of E-cigarettes are less likely to quit smoking than those who have never used it . As early as 19 September 2008, the WHO declared that E-cigarettes were not proven to be effective for nicotine replacement therapies, and there was no scientific evidence that the product was safe and effective . Most importantly, nicotine is a highly addictive substance. If it cannot be strictly and carefully controlled, the function of the E-cigarette as a tool for nicotine replacement for the purposes of smoking cessation will not be fully achieved . E-cigarettes are packaged as harmless smoking-cessation products by sellers with the suggestion that the amount of nicotine in the e-liquid be gradually reduced, which would help smokers get rid of their nicotine dependence and achieve the goal of quitting smoking . However, the nicotine content of some E-cigarettes is not consistent with the labeling . In the process of smoking E-cigarettes, few people are able to get standardized guidance. Generally, the content of nicotine in the e-liquid is added quantitatively. In order to increase the number of users, different manufacturers have added various flavors of spices, many of which have not been approved by the authorities. Therefore, the efficacy of E-cigarettes for smoking cessation requires further research and demonstration.
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review
| 99.9 |
Smoking is a major independent risk factor for cardiovascular diseases (CVD), and the amount of smoking is an independent risk factor for mortality in patients with coronary atherosclerosis. The total amount of cigarette smoking is significantly associated with coronary artery disease and coronary artery stenosis. There is also evidence showing that either active or passive smoking can constitute a risk factor for cardiovascular disease. Passive smoking increases the risk of CVD by 70% to 80%, and this result is similar to active smoking. The relationship between smoking and cardiovascular disease risk has been confirmed, but the exact mechanism is not clear. The mechanism of atherosclerosis induced by smoking may be related to endothelial cell injury and elevated levels of nitric oxide in the blood. Inflammatory response plays an important role in the occurrence and development of atherosclerosis induced by smoking. It was found that the number of inflammatory cells (such as neutrophils and monocytes) increased significantly in the blood of smokers . Nicotine, as the main harmful component in tobacco, plays an important role in the occurrence and development of atherosclerosis . Although the e-liquid has removed tar and other harmful ingredients, more work needs to be done to evaluate its safety due to the existence of nicotine and other potential hazards such as aerosol particles and different flavor additives in e-liquid.(1)The harm of nicotine: smoking has become an independent risk factor for the formation of atherosclerosis, and the addictiveness of nicotine is known to all. Nicotine induces the release of catecholamine and cortisol, and causes hemodynamic changes (increase in heart rate, rise in blood pressure, and the vasoconstriction) and adverse effects on blood lipids (leading to activation of adenylate cyclase in adipose tissue and decomposition of triglycerides. The study found that the total lipid composition of the rats exposed to E-cigarettes smoke increased significantly, the content of saturated fatty acids increased significantly, while the content of unsaturated fatty acids decreased significantly), as well as the induction of insulin resistance . Nicotine can also cause endothelial dysfunction, inhibit apoptosis, and enhance angiogenesis. This effect raises concerns with regard to nicotine promoting cancer development and accelerating atherosclerosis . Researchers at Danderyd Hospital in Sweden have found that inhaling E-cigarettes only 10 times can cause signs of damage to the blood vessels. Subsequently, they further studied the effects on healthy people of inhaling E-cigarettes 30 times. Magnus Rudbeck, a doctor at Danderyd Hospital who participated in the study, believes that E-cigarette users have poor vascular elasticity, and the poor vascular elasticity may lead to heart disease and stroke. Researchers believe that the nicotine in E-cigarettes may lead to poor vascular elasticity . The study of Flouris, A.D. et al. found that white blood cell count increased after smoking E-cigarettes, which reflected the inflammatory process of acute cardiovascular events . The health status of E-cigarette users was analyzed. It was found that, compared with non-smokers, the former had an enhancement in cardiac sympathetic nerve excitability, and they were more prone to oxidative stress reactions. Professor Choupo Perk from European Society of Cardiology explained that the stimulation of atomized nicotine on sympathetic nerves can cause irregular beating of the heart and elevated blood pressure, and may cause long-term harmful effects on the growth of the vascular wall . Vlachopoulos, C. et al. investigated cardiovascular risk factors among 24 young smokers in 4 different smoking scenarios, and used femoral artery to femoral pulse wave velocity (PWV) to assess aortic stiffness, finding that E-cigarette smoke increased young people’s arterial stiffness and blood pressure, and smoking E-cigarettes for over 30 min had an adverse effect on arterial stiffness that was similar to that of traditional cigarettes . The study of Battista, L. et al. also showed that nicotine inhalation vapors produced the same pathophysiological cardiovascular effects as traditional cigarettes . Farsalinos, K.E. et al. studied the acute effects of E-cigarette smoke on the cardiovascular system of exposed people, and the results showed that the cardiac output blood pressure of people exposed to E-cigarettes (11 mg/mL of nicotine content) increased slightly; when the heart of smokers contracted, cardiac output and heart rate were significantly increased. They also used the MTT test to authenticate that the atomized e-liquid has toxic effect on cultured myocardial cells .(2)The hazards of aerosol particles were also studied. Zhang, Y. et al. showed that 73~80% of the particles were in exhaled aerosols, while 7~18% of the particles were stored in the alveoli. It is estimated that 20~27% of the particles entered the circulatory system, which is equivalent to the proportion for traditional cigarette smoke . Moreover, the size distribution and amount of particles of E-cigarettes are similar to those of traditional cigarettes, and some E-cigarettes produce more particles than traditional cigarettes. Particles may cause lung and systemic inflammation, and increase the risk of cardiovascular disease, respiratory disease and death .
|
review
| 99.9 |
The harm of nicotine: smoking has become an independent risk factor for the formation of atherosclerosis, and the addictiveness of nicotine is known to all. Nicotine induces the release of catecholamine and cortisol, and causes hemodynamic changes (increase in heart rate, rise in blood pressure, and the vasoconstriction) and adverse effects on blood lipids (leading to activation of adenylate cyclase in adipose tissue and decomposition of triglycerides. The study found that the total lipid composition of the rats exposed to E-cigarettes smoke increased significantly, the content of saturated fatty acids increased significantly, while the content of unsaturated fatty acids decreased significantly), as well as the induction of insulin resistance . Nicotine can also cause endothelial dysfunction, inhibit apoptosis, and enhance angiogenesis. This effect raises concerns with regard to nicotine promoting cancer development and accelerating atherosclerosis . Researchers at Danderyd Hospital in Sweden have found that inhaling E-cigarettes only 10 times can cause signs of damage to the blood vessels. Subsequently, they further studied the effects on healthy people of inhaling E-cigarettes 30 times. Magnus Rudbeck, a doctor at Danderyd Hospital who participated in the study, believes that E-cigarette users have poor vascular elasticity, and the poor vascular elasticity may lead to heart disease and stroke. Researchers believe that the nicotine in E-cigarettes may lead to poor vascular elasticity . The study of Flouris, A.D. et al. found that white blood cell count increased after smoking E-cigarettes, which reflected the inflammatory process of acute cardiovascular events . The health status of E-cigarette users was analyzed. It was found that, compared with non-smokers, the former had an enhancement in cardiac sympathetic nerve excitability, and they were more prone to oxidative stress reactions. Professor Choupo Perk from European Society of Cardiology explained that the stimulation of atomized nicotine on sympathetic nerves can cause irregular beating of the heart and elevated blood pressure, and may cause long-term harmful effects on the growth of the vascular wall . Vlachopoulos, C. et al. investigated cardiovascular risk factors among 24 young smokers in 4 different smoking scenarios, and used femoral artery to femoral pulse wave velocity (PWV) to assess aortic stiffness, finding that E-cigarette smoke increased young people’s arterial stiffness and blood pressure, and smoking E-cigarettes for over 30 min had an adverse effect on arterial stiffness that was similar to that of traditional cigarettes . The study of Battista, L. et al. also showed that nicotine inhalation vapors produced the same pathophysiological cardiovascular effects as traditional cigarettes . Farsalinos, K.E. et al. studied the acute effects of E-cigarette smoke on the cardiovascular system of exposed people, and the results showed that the cardiac output blood pressure of people exposed to E-cigarettes (11 mg/mL of nicotine content) increased slightly; when the heart of smokers contracted, cardiac output and heart rate were significantly increased. They also used the MTT test to authenticate that the atomized e-liquid has toxic effect on cultured myocardial cells .
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review
| 99.9 |
The hazards of aerosol particles were also studied. Zhang, Y. et al. showed that 73~80% of the particles were in exhaled aerosols, while 7~18% of the particles were stored in the alveoli. It is estimated that 20~27% of the particles entered the circulatory system, which is equivalent to the proportion for traditional cigarette smoke . Moreover, the size distribution and amount of particles of E-cigarettes are similar to those of traditional cigarettes, and some E-cigarettes produce more particles than traditional cigarettes. Particles may cause lung and systemic inflammation, and increase the risk of cardiovascular disease, respiratory disease and death .
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study
| 99.9 |
Traditional cigarette products have been used for hundreds of years. Although cigarette products have been favored by a considerable number of people, the potential hazards caused by smoking have also been paid a great deal of attention. Under these circumstances, governments have launched a series of policies and regulations to control the use of tobacco. With people paying more and more attention to their health, smoking cessation has been widely considered and accepted by the public. On the other hand, tobacco products are now being developed with greater diversification, and can be smokeless. One important form of new tobacco product, E-cigarette products, have been developed, and have quickly dominated the market share. E-cigarettes don’t burn or produce smoke and don’t contain solid particles or tar, while traditional smoke produces these harmful chemicals. Therefore, E-cigarettes are often considered to be safe, and are allowed to be used in public places in some countries and regions. Since the emergence of E-cigarettes, a number of scientists have done a lot of research to investigate their safety. Nevertheless, there is still no specific conclusion. The variation in the classification and control policies relating to the sale and production of E-cigarettes in different countries makes it more difficult to evaluate their safety. Although E-cigarettes only entered the market a short time ago, their characteristics of being both smokeless and relatively healthy for the environment and for health have led them to be quickly accepted and consumed by the public. At present, the main form of E-cigarette is the renewable liquid type. Due to its being fashionable, economical and practical, this is still the future focus of E-cigarettes in the next decades. Furthermore, different amounts of nicotine are added and used in E-cigarettes in various countries, which counteracts the principles of nicotine replacement therapy advocated by the WHO. Actually, there is no solid evidence showing that smoking cessation could be achieved by using E-cigarettes.
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review
| 99.9 |
It is only ten years since E-cigarettes were first developed as a commercial product. E-cigarettes are consumed by inhalation through the respiratory tract and air exchange with lung. So it is not clear if E-cigarettes cause damage to the respiratory tract and other parts of the body. Additionally, the small sample sizes in the population survey and the epidemiological statistics analysis have limited the study of their safety. Under the current circumstances, it is of great importance to investigate the influence of E-cigarettes on human health. Furthermore, more information needs to be provided to the public in terms of guidance for the safe and reasonable use of E-cigarettes. For example, whether or not addiction will be induced after long-term use of E-cigarettes is a question that should be paid more attention. The safety of the equipment is another concern. Meanwhile, specific policies should be authorized by governments to guarantee the quality of production process, the specifications of the E-cigarette, and its safety evaluation. The current review provides relevant information useful for understanding the constitution, history and safety evaluation of E-cigarettes based on the existing literature.
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review
| 99.9 |
The harm of E-cigarettes cannot be underestimated. In vivo and in vitro evidence has confirmed that the components contained are harmful to the respiratory system and the cardiovascular system. Moreover, the levels of harmful components, which include volatile organic compounds, tobacco-specific nitrosamines, and heavy metals, in E-cigarettes are higher than those in traditional cigarettes. At present, the research on E-cigarettes is fragmentary and incomplete, and there has been no systematic review of its safety. Additionally, the lack in quality control and authorized policies for E-cigarettes makes it inconvenient to conduct further research and development.
|
review
| 99.9 |
Our genome integrity is under attack every day by a variety of exogenous and endogenous sources . Multiple cellular DNA repair mechanisms exist to remove damaged regions of chromosomes . Besides DNA repair, the genetic information can also be protected by other biological processes such as cell cycle checkpoints and apoptosis . Depending on the damage sources, DNA encounters diverse types of lesions such as base modifications, single strand breaks (SSB), or double strand breaks (DSB) that perturb the primary structure of DNA . Among DNA damage, DSBs are the most deleterious DNA lesions in light of their high levels of propensity to evoke genomic instability and cancer . Homologous recombination (HR) and nonhomologous end joining (NHEJ) are the two major pathways responsible for the repair of DSBs in higher eukaryotes. Mutations in DSB repair proteins are frequently associated with an increased risk of cancer . Besides, hyperactivation of DSB repair genes is one of the reasons for radio- and chemoresistance . In addition to understanding the critical roles of protein-coding driver genes in DNA damage response (DDR), efforts have been focused on identifying long non-coding RNAs (lncRNAs) that are largely transcribed from cancer risk loci and investigating how they can be potential biomarkers during anti-cancer therapy . LncRNAs are deregulated in cancer tissues and their altered expressions are most likely caused by copy number variations or single nucleotide polymorphisms (SNPs) [8, 9]. Given that the majority of lncRNAs have no assigned function, they are likely to provide an abundance of opportunities for revealing novel pathways that could conceivably be targeted for cancer therapy. Throughout this review, we guide readers to the most recent studies that describe in great detail unique characteristics of lncRNAs during DSB repair pathways. We first define the link between DSB and tumorigenesis and then describe noteworthy anti-cancer regimens using DSB repair. Finally, we focus on reviewing the functional roles of lncRNAs in DDR.
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review
| 99.9 |
DNA double strand breaks are among the most detrimental damages which can lead to severe genome rearrangements. The two main repair pathways triggered by DSBs are homologous recombination and nonhomologous end joining repair . It has been proposed that chromatin state and damaged positions determine which pathway is favored [11, 12]. The error-free homologous recombination repair is a multistep procedure containing three main steps: initially (presynaptic phase), DSB is recognized and processed to give a 3’ single-stranded overhang by the MRE11-RAD50-NBS1 (MRN) complex . This 5′–3′ DNA end resection is reinforced by replication protein A (RPA) . Next (synaptic phase), DNA strand invasion takes place when RAD51 binds to single stranded DNA and displaces RPA, which leads to RAD51 polymerization. RAD52 and p53 can control this process . After the homology search, the heteroduplex structure is formed and stabilized by RAD54/p53 complex . Finally (postsynaptic phase), DNA polymerases use the intact sister chromatid strand to re-synthesize fragments and the Holliday junctions are resolved by specific endonucleases that are called as resolvases .
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study
| 99.94 |
When the homologous template is unavailable, the break ends are directly ligated through nonhomologous end joining repair. The initial step in NHEJ repair is the recognition and binding of the Ku70/80 heterodimer to the DSB . Subsequently, Ku serves as a scaffold to bring the other NHEJ factors to the damage site, including DNA-PKcs, XRCC4, DNA ligase IV, XRCC4-like factor (XLF) and/or ATM and ATR . Interestingly, the order of the sequential recruitment of these factors to the DSB mediated by the Ku heterodimer is quite flexible . Upon binding, Ku-DNA-PKcs or XRCC-XLF can bridge and stabilize DSB ends by protecting them from non-specific processing which may lead to chromosome aberrations. The next step is to make the ends ligatable by removing the damaged or mismatched nucleotides at DSB ends. Many enzymes, including PNKP, Artemis and Ku itself, have been reported responsible for processing DNA ends for the NHEJ pathway [21–23]. The ultimate step in NHEJ is gap filling by DNA polymerase and ligation of the broken ends by DNA Ligase IV whose activity can be stimulated by XRCC4 .
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study
| 99.94 |
DNA double-strand break repair pathways have a multifaceted function in tumorigenesis and in the response to therapeutic modalities. Firstly, erroneous or deregulated DNA repair results in chromosomal abnormalities, genomic instability, and higher mutation rates, which can predispose the cells to cancer and make them vulnerable to certain kinds of genotoxic stresses . On the other hand, hyperactive DNA repair proteins due to upregulation or polymorphisms may provide survival advantages to cancer cells in therapeutic response . Defects in core HR and NHEJ proteins have been implicated in a vast repertoire of cancers (Table 1). It has been estimated that approximately half of high-grade serous ovarian adenocarcinoma samples are defective in HR repair pathway, and these HR defects are largely driven by mutations or epigenetic silencing of BRCA1 and BRCA2 genes . In terms of the major cellular sources of DSBs, evidence has shown that the DDR can be invoked and dysfunctional at an early stage in the development of neoplasia [28, 29]. The activation of oncogenes, for example, MYC and RAS can stimulate the firing of various unwanted replication forks as a major aspect of a proliferative program. These forks rapidly stall and collapse, resulting in formation of DSBs [30, 31]. Cell-cycle checkpoints are elicited to repair DNA lesions before mitosis takes place. For precancerous damage to advance to tumors, it is suggested that DSB repair factors and cell-cycle checkpoint proteins progress toward becoming inactivated. Thus, cells continue through the cell cycle with unsuccessfully repaired collapsed forks in place, resulting in tumor growth and expansion . Additionally, there is strong association between DSB repair gene mutations and an elevated risk of inherited rare diseases. Mutations in ATM, Mre11 and NBS1 are found in patients with Ataxia Telangiectasia (A-T), Ataxia Telangiectasia-like disorder (A-TLD) and Nijmegen breakage syndrome (NBS), respectively .
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review
| 99.9 |
Radiation therapy which causes a variety of DNA lesions including DSBs damage continues to be a mainstay in the treatment of an assortment of malignancies . However, tumor cells as seen in many cancers often display resistance to standardized radiation therapy due to hyperactive DSB repair mechanisms . Therefore, developing drugs aimed at modulating DSB repair activity has provided a profound avenue for many commonly used chemotherapy and radiotherapy regimens. One of the well-known cases is the utilization of platinum salts which is frequently given in patients with advanced ovarian cancer . Platinum salts (carboplatin or cisplatin) can cause DNA inter- and intrastrand crosslinks damages that are recognized and repaired by a combination of nucleotide excision repair (NER) and HR . It has been estimated that nearly half of high-grade serous ovarian cancers have germ line or somatic mutations in BRCA1 or BRCA2 [38, 39]. Many DSB repair proteins are now being used as biomarkers to direct the use of therapy (Table 2). Although tumor cells defective in the repair genes show sensitivity toward genotoxic agents in the first place, after an unpredictable period, hyperactivity of the repair proteins due to re-emergence, reversal or overlapping compensatory pathways can make cancer cells resistant and account for the relapse . This is especially true also in ovarian cancer, in which more than 42% of the carboplatin-resistant tumors tested had secondary mutations that restored the BRCA1 or BRCA2 open reading frames . Tumor cells are highly heterogenous and have the ability to develop either intrinsic or acquired resistance phenotype through molecular alterations. This poses a major challenge to cancer treatment. One possible way to overcome or delay the development of chemoresistance is to ‘re-sensitize’ tumors to the original treatment with the help of new identified targets.
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review
| 99.9 |
Besides DSB repair proteins, mutations in long non-coding RNAs (lncRNAs) are associated with tumorigenesis. Large scale screening has provided novel p53 interactors, including lncRNAs, which can be potential therapeutic targets [41–43]. Researchers have identified 22 distinct lncRNAs that are involved in the regulation of chemoresistance in cancers . How the lncRNA regulatory networks act in concert to modulate oncogenesis and therapeutic response remains largely unknown. Here we review the most recent findings on lncRNAs with well-characterized functions in DDR.
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review
| 99.9 |
The fast-growing RNA-seq technique has found that distinct sets of lncRNAs are expressed in correlation with different physiological and pathological cellular processes . The earliest attempts to elucidate the function of thousands of lncRNAs with highly conserved chromatin signatures in specific biological pathways have shown that 39 lncRNAs were significantly induced in p53-mediated DNA damage response . Most recently, Howard Chang and colleagues performed a screen for transcribed regions around the promoters of cell cycle genes and discovered a new regulatory lncRNA – DINO (damage-induced non-coding RNA), expanding the p53 network (Figure 1A) [41, 47]. DINO is transcribed divergently from CDKN1A (p21) promoter, with ~100-fold increase upon doxorubicin treatment in a p53-dependent manner . In terms of the functions of DINO in the p53-mediated DNA damage pathway, the authors have found that DINO can physically interact with the C-terminal RNA-binding region of p53 and colocalize at multiple p53 target genes including CDKN1A throughout the genome to co-regulate the p53-dependent gene expression and cell cycle arrest in response to DNA damage. More importantly, the microhomology region of DINO interacting with p53 is highly conserved in mammal species, although DINO exhibits poor overall sequence identity across species, suggesting that DINO represent a conserved transcriptional response after DNA damage . To further discern whether the effects observed are due to disruption of the DINO transcripts or the DNA binding platform for other regulatory factors, the study presented two different transgenic knockout mouse models in which the promoter of Dino is either intact or inactivated. Both lines of Dino knockout mice exhibit impaired response to doxorubicin, suggesting that in mouse Dino acts in trans similarly to human DINO . This study identified a new lncRNA that constitutes a feed-forward feedback loop in the p53-dependent DNA damage response. However, it remains unclear how DINO stabilizes p53 and whether DINO mutations are putatively correlated with cancer diagnosis and prognosis.
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study
| 89.06 |
(A) DINO is increased upon DNA damage in a p53-dependent manner and physically interacts with p53, resulting in p53 stabilization and activation of p53 target genes cell cycle arrest. (B) TP53TG1 is stimulated by p53 upon DNA damage and binds to the DNA/RNA binding protein YBX1 to prevent its activation of oncogenes. TP53TG1 inactivation by methylation in cancer cells releases the transcriptional repression of YBX1-targeted growth-promoting genes.
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study
| 100.0 |
Innate and acquired chemoresistance exhibited by most tumors exposed to conventional chemotherapeutic agents account for most relapse cases in cancer patients . In addition to multiple key well-known molecular players, lncRNAs have been shown to be involved in the development of chemoresistance . Most recently, Manel Esteller's group has discovered the lncRNA – TP53TG1 as a chemo-sensitizer to promote p53 response to DNA damage (Figure 1B) . By comparing DNA methylation profiles of colon cancer cell line HCT-116 with or without disrupted DNA methyltransferase enzymes, along with normal colon cell line, the authors have identified a dozen of lncRNAs which exhibited CpG island hypermethylation-associated transcriptional silencing in colon cancer cells. In normal colon tissues, the p53 protein binds the regulatory region of the TP53TG1 molecule and activates it in response to cellular stress . Thereafter, TP53TG1 blocks activation of the YBX1 protein that, when induced, goes into the cell nucleus and stimulates many oncogenes . 10% of colon and stomach tumors show inactivation of the TP53TG1 molecule due to hypermethylation at CpG island, furthermore, oncology patients with inactive TP53TG1 have a shorter progression-free survival . TP53TG1 silencing in cancer cells causes the p53 protein to lose its antitumor effects and free the RNA binding protein YBX1 to activate oncogenes that prevent the death of malignant cells in response to anti-tumor drugs, resulting in chemoresistance . Of course, YBX1 should not be the only ‘hit’ resulting from the epigenetic loss of TP53TG1 in the center p53 link. Alternative targets would warrant further research. Esteller's lab and others have underscored that epigenetic factors are highly associated with multi-resistance of tumors to most common drugs, although it remains unclear how these epigenetic mutations occur in tumor cells.
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review
| 99.8 |
Given the vital role p53 plays in cancer, efforts have focused on finding a means of restoring functional p53 in human cancer cells. Normally lncRNAs exert a diverse spectrum of regulatory mechanisms, a special lncRNA NEAT1 (nuclear-enriched autosomal transcript) is mainly localized to nuclear paraspeckles, subnuclear particles that can be found in the cell nuclei of cancer cells . A recent study has illustrated that targeting NEAT1 and ‘paraspeckles’ would be a new therapeutic avenue in the fight against cancer (Figure 2A) . The authors have observed that NEAT1 is increased in Nutlin-3a-treated p53 wild type cancer cell lines. Strikingly, although NEAT1 is regulated by p53, it is required for the survival of highly dividing cancer initiating cells and that mice lacking NEAT1 are protected from developing skin cancer . NEAT1 depleted cancer cells exhibited a much higher level of γH2A.X accumulation and this higher amount of DNA damage was exacerbated in response to replication stress . These surprising results suggest that cancer cells can ‘hijack’ the survival principle of NEAT1 for their own good. However, there is still a long way to go before the information can be harnessed to help cure cancer. For instance, how exactly NEAT1 confers its survival functions to cells is worth further investigation. Interestingly, the most recent study has found that some NEAT1 isoforms reside in numerous non-paraspeckle foci and exert distinct functions . Therefore, precise disruption of NEAT1 isoforms via genome editing tools is demanded in clinical trials for targeting overlapping transcripts.
|
review
| 99.8 |
(A) NEAT1, mainly localized in paraspeckles, is induced by p53 after DNA damage, the paraspeckles with increased NEAT1 then regulates the ATR-mediated DSB repair. (B) At early stage of DSB repair, DDSR1 interacts with BRCA1-RAP80 and hnRNPUL1 to prevent them from promiscuous DNA binding; at late stage of DSB repair, DDSR1 is induced by ATM and NF-κB signaling pathways to ensure efficient repair.
|
study
| 100.0 |
The non-homologous end-joining (NHEJ) pathway and homologous recombination (HR) pathway share the duty to safeguard the genome stability when the most toxic DNA double-strand breaks occur. It is a crucial decision for cells to choose which pathway to orchestrate, in that making a wrong choice can lead to detrimental repair outcomes . Therefore, efforts are taken to identify the cellular determinants involved in the regulation of these two pathways. In a recent study, Misteli and colleagues treated immortalized human fibroblasts with DSB-inducing agents and found a lncRNA named DDSR1 (DNA damage-sensitive RNA1) which was highly induced upon damage in an ATM and NF-κB dependent manner (Figure 2B) . Interestingly, p53 was not required for DDSR1 induction after DNA damage, but DDSR1 can largely regulate p53 target genes under stress conditions. Moreover, cell proliferation and DNA damage signaling were reduced in cells lacking DDSR1 . In order to investigate the underlying mechanism by which DDSR1 affects DNA repair, the authors applied DSB repair reporter cell lines and found that only the HR pathway was compromised in DDSR1-deficient cells. The authors also found a RNA binding protein hnRNPUL1 physically associating with DDSR1. HnRNPUL1 has been reported to promote DNA end resection in HR pathway . Thus, further analysis demonstrated that depletion of DDSR1 increased accumulation of BRCA1/RAP80 complex at sites of DNA damage to restrict DNA end resection . Given that induction of DDSR1 took several hours to occur, while recruitment of BRCA1/RAP80 to DNA damage sites happens within several minutes, DDSR1 could possibly have dual roles in regulating HR: at early stage, DDSR1 associates with BRCA1/RAP80 complex to prevent them binding to damaged chromatin. Subsequently, DDSR1 expression is increased by ATM and NF-κB to inhibit p53 target gene expression . This study highlights the multifaceted nature of lncRNAs in maintaining genome integrity, providing new insights onto the precise targeted therapy for cancer.
|
review
| 97.8 |
It is always a big challenge to interpret the mechanisms of action of risk-associated SNPs from GWAS analysis, given that more and more SNPs are found to lie in non-coding regions of the genome. Most recently, researchers have identified two novel lncRNAs that were transcribed in the proximal cancer risk loci and characterized the important roles in tumorigenesis (Figure 3) . Previously it's been found by the same group that the strongest risk-associated SNPs fall within the enhancer region named PRE1 that regulates the expression of CCND1 . PRE1 also acts as an enhancer on the lncRNAs CUPID1 and CUPID2 which are transcribed from a bidirectional promoter. The two lncRNAs were highly expressed in breast cancer cell lines dependent on estrogen . However, this induction was not associated with PRE1 region amplification, indicating that copy-number variation is not the only mechanism underlying the expression. Unlike CCND1, silencing of CUPID1 or CUPID2 did not affect cell cycle, although depletion of these two lncRNAs indeed caused deregulation of DNA replication, recombination and repair genes . The authors have illustrated that CUPID1 and CUPID2 can facilitate the formation of phosphorylated RPA foci and promote RAD51 recruitment to DSBs during the initiation step of the HR pathway. Interestingly, when breast cancer risk SNPs were incorporated into PRE1 region, leading to the decreased expression of these two lncRNAs, the overall DSB repair was not impeded, but a large number of structural variants across the genome were observed . These data proposed a clear regulatory role for these two lncRNAs: CUPID1 and CUPID2 prevent the breast tumors from error introduction in response to radiotherapy by favoring a switch from NHEJ to HR DSB repair.
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study
| 99.94 |
The discovery of lncRNAs has dramatically changed the understanding of the biology of human diseases, especially when genomic studies of tough-to-treat cancers have mainly focused on protein-coding genes and provided no effective targeted therapies. Many TNBC (triple negative breast cancer) patients poorly respond to chemotherapy and radiotherapy due to EGFR (epidermal growth factor receptor) gene amplification and TP53 mutations [60, 61]. Most recently, researchers have identified a lncRNA – LINP1 – that regulates the sensitivity of the tumor cells to radiation therapy (Figure 4A) . LINP1 was initially identified as overexpressed in TNBC when compared with other breast cancer subtypes using RNA-seq data from the Cancer Genome Atlas and the Cancer Cell Line Encyclopedia. Among the dozens of lncRNA candidates, LINP1 stands out as functional screening has revealed that LINP1 knockdown enhanced apoptosis in TNBC cell lines following doxorubicin treatment (a chemotherapy drug for TNBC) . By applying in-vitro synthesized and also endogenous RNA, the authors demonstrated that LINP1 transcript physically interacted with Ku80-DNA-PKcs complex. Therefore, LINP1 knockdown in TNBC cells led to reduced DSB repair, and conversely, overexpression of LINP1 in ER-positive cells increased NHEJ activity . The authors uncovered that EGFR activation upregulates LINP1 transcription, thus in turn stabilizes the Ku80-DNA-PKcs interaction. On the other hand, TP53 activation stimulates miR-29 that targets LINP1 and down-regulates its expression later point after damage. Thus, TP53 mutations in TNBC would further increase LINP1 expression at the post-transcriptional levels after DNA damage. Given that inhibition of the NHEJ pathway has been proposed by oncology researchers to synergize DNA-damaging therapies for better treatment outcomes for TNBC, LINP1, as a new class of cancer-driver gene that links two repair scaffold proteins, may serve as a novel therapeutic target for TNBC treatment. While this study mainly focused on TNBC, these findings have left an open question: to what extent cellular LINP1 expression levels indicate NHEJ functional status, since overexpression of LINP1 increases resistance to genotoxic insults and loss of expression of LINP1 may impair genome stability.
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review
| 99.7 |
(A) LINP1 is induced by EGFR upon DNA damage and further stabilized by inactivation of miR-29 which is stimulated by p53. LINP1 can physically interact with Ku80-DNA-PKcs complex and promote DSB repair. (B) Short isoform of ASCC3 lncRNA is produced via alternative splicing after DNA damage and facilitates transcription recovery after DNA repair; while the long version of ASCC3 (protein coding transcript) inhibits transcription recovery.
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study
| 100.0 |
Ultraviolet light can damage DNA, triggering a general transient shutdown of gene transcription . This response has been known for a few decades. However, the molecular mechanism underlying transcription shutdown and recovery upon DNA damage is still largely unknown. Also, in contrast to global transcription repression, some genes are activated by UV light . An investigation of this counter-intuitive behavior implies a surprising gene regulation mechanism . The latest studies have focused on identifying novel factors associated with transcription-related changes after UV-induced DNA damage (Figure 4B) . By using next-generation DNA and RNA sequencing technology, the authors have revealed a global switch in pre-mRNA processing resulting in a preference for the production of transcripts containing alternative last exons which are not normally included in the dominant canonical mRNA isoforms. By combining siRNA-mediated functional screening, ASCC3 stands out as a pivotal regulator of transcription following UV damage. Knocking down the short isoform of ASCC3 transcript (functionally a lncRNA) prevented the cells from recovering normal levels of transcription. In contrast, blocking the long isoform of ASCC3 transcript (encoding a full-length protein) increased transcription levels after UV irradiation . This alternative last exon-derived non-coding RNA produced from a protein-coding gene provides a new source of lncRNAs. Too much exposure to UV radiation is the main cause of skin cancer development. A clearer understanding of UV-induced DNA damage repair is crucial in the prevention of skin cancer.
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review
| 99.6 |
The simplest definition for lncRNAs is RNA genes larger than 200 base-pairs that do not appear to have coding potential. However, the characteristics of lncRNAs are far more complex than were originally imagined, as they are involved in numerous biological processes across many aspects of life rather than just results of transcriptional noise. In normal proliferating cells, lncRNAs are expressed, on average, at much lower levels than coding genes [67, 68]. During DNA damage response, many lncRNAs are dramatically induced. With current deep sequencing technology, the rate of discovering new lncRNA genes is rapidly overwhelming the rate of characterizing them. The hurdles to characterize lncRNAs are not only due to experimental challenges, but also and more importantly due to ambiguous results from only RNA-seq experiments, as for most lncRNAs, the action of transcription alone is sufficient for their function but the transcript itself is not necessary [69–71]. To discover more functional lncRNAs during cellular response, efforts are still needed to apply multiple powerful approaches. Firstly, given that the biogenesis and processing of lncRNAs is quite distinguished from mRNAs in normal proliferating cells , it is likely that the transcriptional profiles of lncRNAs in response to DNA damage have unique features. mNET-seq (mammalian native elongating transcript sequencing) can provide critical evidence on the active transcription of lncRNAs under stress conditions . Secondly, visualizing the mobility of DNA repair factors in real time during cellular responses can be achieved by using local irradiation and live cell imaging . lncRNAs with essential roles in DDR (DNA damage response) signaling pathways can also be detected by using these molecular analyses. Thirdly, unbiased genome-wide CRISPR screening will yield a great appreciation for lncRNAs’ biological functions. Jonathan Weissman's and Daniel Lim's labs have developed a CRISPR interference platform that targeted thousands of lncRNA loci and found hundreds of them for robust cell growth in at least one cell type . This modified CRISPR approach can be expanded to pinpoint lncRNA transcripts that are important during DDR. Lastly, unlike mRNAs, lncRNAs are mostly restricted to nucleus and ~60% of annotated lncRNAs are chromatin-enriched , and they are poorly co-transcriptionally processed and are rapidly degraded by the RNA exosome . Due to diverse functions of lncRNAs in various biological phenomena, it would be important to study the localization of these molecules during DDR.
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review
| 99.9 |
LncRNAs can have pro-survival or pro-apoptotic functions in response to DNA damage that can be utilized for future translational research. Given that expressions and functions of lncRNAs are highly cell type specific, cancer treatments may benefit from targeting lncRNAs crucial to cancer cell function, whereas having little effect on nearby normal cells that do not require these lncRNAs. The connection between DSB repair and ionizing radiation has been solidly established and work to date proposes that targeting the DSB repair pathways still has extensive potential for expanding radio- and chemosensitization in the clinic . Although there have been no efforts so far to develop drugs targeting lncRNAs in a clinical setting, it is encouraging to see that more and more lncRNAs have been identified with well-defined functions, these findings demonstrate that the therapeutic potential of lncRNAs warrants further investigation.
|
review
| 99.9 |
In the past decades, the Internet played an important role in our life. However, more and more adolescents surf the Internet and play online game excessively, which result in adverse effects on adolescents themselves and society. An epidemiological study demonstrated that Internet gaming disorder (IGD), a subtype of Internet addiction (IA) (1), was a very common mental health problem among Chinese adolescents (2). Therefore, more and more studies focused on the neuromechanism of IGD and aimed to contribute to the prevention and treatment of IGD.
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review
| 98.1 |
Structural neuroimaging of brain could be used to investigate brain mechanisms about individual personality traits (3–5). Previous structural studies have found that individuals with IGD had structural abnormalities in gray matter (GM), such as decreased gray-matter volume (GMV) or GM density in multiple cortical and subcortical areas (6–11), and increased GMV in frontal and temporal regions (8, 12). These studies suggested that multiple brain areas in the frontal, temporal, parietal, and subcortical regions such as ventral striatum were associated with IA, which contributed to the understanding of the neuromechanisms of IA. However, the majority of previous studies just focused on the IA or IGD diagnosed by clinical questionnaire such as Internet addiction test (IAT), and compared the differences in behavior and brain function and structure between the IGD individuals and healthy controls. As a matter of fact, not all the individuals who play online game suffer from the IGD (13). Therefore, investigation of the structural correlations in online game players with different levels of tendency to IGD, not only the individuals with IGD diagnosis, is necessary.
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study
| 99.75 |
Recently, three studies directly focused on the neural associations of the tendency to the IA. Wen and Hsieh (14) explored the relationship between the whole brain functional connections and the level of IA in a group of young adults (19–29 years) and found two networks mainly consisted of frontal regions were correlated to the tendency of IA. Li et al. (15) reported that the structure and functional connectivity of the right dorsolateral prefrontal cortex were positively correlated with the IAT score in a group of healthy young adults (18–27 years). A study by Kühn (16) revealed that the GMV of the brain regions within fronto-striatal network correlated to excessive Internet use assessed by IAT score. Additionally, previous studies have also demonstrated that the GMV changes were related to the online game addiction severity in the IGD subjects. For example, a study by Weng et al. demonstrated that the GMVs of the right orbitofrontal cortex and bilateral insula were positively correlated with the online game addiction severity in the IGD subjects (7). Cai et al. reported increased GMV of nucleus accumbens was associated with the IAT score in the IGD individuals (17). A study by Zhou et al. showed that lower GMV in the right orbitofrontal cortex was related to higher online video gaming addiction severity within the Internet gamers (18). These studies demonstrated that brain structures and functions were associated with the level of IA. However, the relationship between the tendency to IGD and the GMV across whole brain was not yet clearly evaluated in adolescents (14–18 years). The adolescent between 14 and 18 years of age is in a critical period of psychological development and is prone to addiction and adverse effects (19, 20). Many studies regarding the substance addiction paid close attention to adolescents aged from 14 to 18 years (21, 22). A large-sample study demonstrated that the IGD is very common in Chinese elementary and middle school students with a incidence of 22.5% among those students who play online games (2). Therefore, it is more necessary to investigate the brain structural correlations with the tendency to IGD in adolescents (14–18 years).
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review
| 99.5 |
Furthermore, previous studies demonstrated that long-term online game playing could lead to structural reorganization of the brain in online game players (12, 23, 24). The GMVs in the ventrolateral prefrontal cortex, the dorsolateral prefrontal cortex, the supplementary motor area, and the rostral anterior cingulate cortex were correlated with the duration of online game playing in the adolescents with IA disorder (6, 25). Therefore, whether the duration of online game playing affects the relation between the GMV and the tendency to IGD is worth studying.
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study
| 100.0 |
In the present study, 67 male adolescents (14–18 years) who played online games were recruited. The voxel-based correlation analysis was conducted to detect the brain regions associated with IAT score before and after controlling for the total time of playing online game. Based on the previous studies, the prefrontal-striatal circuits are closely related to the addiction. Ventral striatum participated in the habit learning and rewarding process involved in addiction (26, 27), and the reduced control effect of prefrontal cortex on rewarding process is one of the mechanisms of addiction (28, 29). Therefore, we hypothesized that the IGD tendency may be associated with the brain regions related to the cognitive control (prefrontal cortex) and the rewarding process (ventral striatum). This study may lead to new targets for preventing and treating the IGD in adolescents.
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study
| 100.0 |
Sixty seven right-handed adolescents (14–18 years old, average 15.54 ± 0.14) who played online game were recruited in this study. Twenty of 67 participants were the students of a Health School and 47 of 67 participants were the adolescents whose parents took them to a psychiatrist because of possible IGD. All participants received education for 6–12 years, ranging from primary school to senior high school. All of the participants spent more than 80% of the online time on playing online game. Only male adolescents were enrolled in this study because relatively small number of females play online games and suffer from IGD (2, 30). Exclusion criteria included the following: alcohol abuse or drug dependence; existence of any neurologic or psychiatric disease such as insomnia, migraines, tinnitus, and attention deficit hyperactive disorder; history of physical illness such as brain trauma, brain tumor, or epilepsy assessed according to clinical evaluations and medical records; MRI contradiction; and visible abnormalities on conventional MRI. The present study was approved by the Ethical Committee of Tianjin Medical University General Hospital, and all of the participants and their guardians provided written informed consent according to institutional guidelines.
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study
| 100.0 |
Internet addiction test was used to assess the severity of the tendency to IGD in this study. The IAT consists of 20 items and the answers of these questions were described as 1–5 score (1 = “rarely” to 5 = “always”) (31). The total score of 20 items measures the severity of Internet dependency. The experience of online game playing was assessed via a self-report questionnaire that questioned about the length and amount of playing. The total time of playing online game was calculated as hours per day multiplied by the days of playing online games. Intelligence Quotient (IQ) of all participants was tested using Standard Raven’s Progressive Matrices. The anxiety and depression were texted by using the self-rating anxiety scale (SAS) and the self-rating depression scale (SDS).
|
study
| 100.0 |
Structural images were obtained using a Siemens 3.0 T scanner (Magnetom Verio, Siemens, Erlangen, Germany). A series of 192 contiguous sagittal high-resolution anatomical images were obtained using a three-dimensional T1-weighted volumetric magnetization-prepared rapid gradient-echo sequence with the following parameters: TR = 2000 ms, TE = 2.34 ms, TI = 900 ms, flip angle = 9°, FOV = 256 mm × 256 mm, slice thickness = 1 mm, matrix size = 256 × 256.
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study
| 99.94 |
All structural images were preprocessed with the VBM8 toolbox1 of the SPM8 (Wellcome Department of Imaging Neuroscience, London, UK)2 running on MATLAB R2010a (Math Works Inc., Sherborn, MA, USA). Three-dimensional geometric correction was performed during reconstructing the images. After that, the individual native images of all participants were segmented into GM, white matter (WM), and cerebral spinal fluid (CSF), and the GM segments were normalized to the Montreal Neurological Institute template by diffeomorphic anatomical registration through exponentiated lie algebra (DARTEL) (32). The registered GM images were then modulated by dividing the Jacobian of the warp field to correct for local expansion or contraction. The isotropic Gaussian kernel of 8-mm full width at half maximum was adopted to smooth the modulated GM images. The mean image of normalized GM from all participants was used to create a GM mask whose threshold was set at a value of 0.3 (pixels with computed GM fraction values >30% were selected). Then the GM mask was used as an explicit mask for the statistical analysis to exclude the pixels with low GM probability values.
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study
| 100.0 |
Voxel-wise multiple regression analysis was carried out to explore the correlation between the GMV and the IAT score across all participants after controlling for the age and years of education. The non-parametric permutation approach (33) was accomplished by the randomize tool commanded in FMRIB Software Library (FSL)3. The threshold-free cluster enhancement (TFCE) analysis was performed as it combines cluster extent and height into one statistic and does not require an arbitrary choice of a cluster forming threshold (34). The correlation between the GMV and the IAT score was assessed using permutation-based non-parametric testing with 5,000 random permutations. The statistical threshold for significance was defined at P < 0.01. For clarifying whether the duration of online game playing affected the correlation between the GMV and the IAT, Voxel-wise multiple regression analysis was conducted again when adding the total time of playing online game as a nuisance covariate.
|
study
| 100.0 |
Clusters with correlation between the GMV and the IAT score were defined as regions of interest (ROIs), and the average GMV within each ROI was extracted. ROI-based correlation analysis was conducted between the average GMV and the IAT score after controlling for the age and years of education. Then, all of the participants were divided into two subgroups, the high IAT score group (IAT score >50, N = 30) and the low IAT score group (IAT score ≤50, N = 37). The difference in the GMV between the two subgroups was tested by General Linear Model analysis, controlling for the age and years of education. The significance levels were both set at P < 0.05.
|
study
| 100.0 |
Voxel-wise correlation analysis revealed that the GMVs of the bilateral postcentral gyri (postCG), the bilateral precentral gyri (preCG), the right precuneus, the left posterior midcingulate cortex (pMCC), the left inferior parietal lobe (IPL), and the right middle frontal gyrus (MFG) were significantly correlated to the IAT score (Figure 1; Table 2). Figure 2 shows the ROI-based correlations between the GMV and the IAT score. After the total time of playing online game was added as a nuisance covariate, the correlation still existed between the IAT and the GMV of the bilateral postCG, the left preCG, the left pMCC, and the right MFG (Figure 3; Table 3).
|
study
| 100.0 |
Brain regions showing negative structural correlates to Internet addiction test (IAT) score in adolescent online game players. The IAT score was negatively correlated to the gray-matter volumes (GMVs) of the bilateral postcentral gyri, the bilateral precentral gyri, the right precuneus, the left posterior mid cingulate cortex, the left inferior parietal lobule, and the right middle frontal gyrus. The numbers below the images are the Montreal Neurological Institute coordinates at z-axis. The colorbar represents the −log p.
|
study
| 100.0 |
Brain regions showing negative structural correlates to Internet addiction test (IAT) score in adolescent online game players after controlling for the total time of playing online game. The IAT score was negatively correlated to the gray-matter volumes (GMVs) of the bilateral postcentral gyri, the left precentral gyrus, the left posterior mid cingulate cortex, and the right middle frontal gyrus. The numbers below the images are the Montreal Neurological Institute coordinates at z-axis. The colorbar represents the −log p.
|
study
| 100.0 |
As seen in Table 4, when the participants were divided into the two subgroups according to the IAT score, the subgroup with high IAT score (IAT score >50) had lower GMV in the seven of eight regions compared with the subgroup with low IAT score group (IAT score ≤50) (P < 0.05).
|
study
| 100.0 |
In the present study, the association between the GMV and IGD tendency was evaluated within the whole brain in adolescent online game players. After controlling for the effect of the total time of playing online game, the GMVs of the bilateral postCG, the left preCG, the left pMCC, and the right MFG were still negatively correlated to the IGD tendency. The adolescents with lower GMV in the brain regions related to sensorimotor process and cognitive control had higher IGD tendency.
|
study
| 100.0 |
It was consistent with the hypothesis that the GMV in MFG, as a part of prefrontal cortex involved in cognitive controls (35, 36), was negatively correlated with the IGD tendency. Structural and functional abnormalities were widely reported in individuals with IGD (37–40). For example, less activation in the prefrontal cortex was found in the IA (40). Previous studies demonstrated the lower GM density and GMV in the prefrontal cortex in the IGD individuals (37, 39). Smaller amplitude of low-frequency fluctuation within the right MFG was also revealed in the IGD individuals (41). Abnormal activation in the prefrontal cortex was also found in drug-addicted individuals such as the marijuana users and the abstinent cocaine abusers (42–44). Similar changes in functional connectivity of the prefrontal cortex were revealed in the individuals with alcohol dependence and the individuals with IGD (45, 46). These studies demonstrated that the structural or functional condition of prefrontal cortex was associated with the addiction. In this study, the GMV of the right MFG was negatively correlated to the IAT score, and was lower in the high IAT score subgroup than that in the low IAT score subgroup. Structural abnormality in the right MFG might lead to the impairment of cognitive control in online game players. As a result, the online game players could not control their problematic online game playing and exhibited a higher tendency to the IGD.
|
study
| 99.94 |
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