O-methylation, 2′-OMe) occurs at high frequencies in rRNAs and other small RNAs and is carried out using a shared mechanism across eukaryotes and archaea. As RNA modifications are important for ribosome maturation, and alterations in these modifications are associated with cellular defects and diseases, it is important to characterize the landscape of 2′-O-methylation. Here we report the development of a highly sensitive and accurate method for ribose methylation detection using next-generation sequencing. A key feature of this method is the generation of RNA fragments with random 3′-ends, followed by periodate oxidation of all molecules terminating in 2′,3′-OH groups. This allows only RNAs harboring 2′-OMe groups at their 3′-ends to be sequenced. Although currently requiring microgram amounts of starting material, this method is robust for the analysis of rRNAs even at low sequencing depth.Ribose methylation (2′- O-methylations are directed by Box C/D snoRNAs, noncoding RNAs that guide the modification of target sites via complementary RNA sequences. In humans, snoRNAs are assembled into snoRNP particles, containing the conserved core proteins NOP56, NOP58, fibrillarin , and 15.5K , thus preventing them from being ligated to linkers for sequencing library construction . As a consequence, the nearest possible position for a 2′-O-methylated base is one base upstream of the original 3′-end of an RNA fragment  is used. Although less efficient than other widely used phosphatases, the advantage is that this enzyme nevertheless is capable of removing all three types of phosphates under acidic conditions in the absence of ATP . The filters were set to correspond to the known sites that have the lowest log2 fold changes and the highest P-values to allow maximum sensitivity . The number of high confidence sites consisted of 93.3% known sites, which include sites newly found and MS validated by 2 fold change of >6 and adjusted P-value remained unchanged, eight total potential novel sites were identified (2 fold change (∼10) and a low adjusted P-value (∼1.5 × 10−13), in 28S was validated using a primer extension under restricted dNTP concentration  cannot cleave at the 3′-site of 2′-OMe, so the 5′-site can never be exposed using our approach. Thus, if G4498 were fully methylated, U4497 would not be detected. Indeed, we did not see U4497, indicating G4498 may be fully methylated. However, another set of back-to-back pairs of 28S sites (U4226 and G4227) were both detected with high confidence. The most probable explanation for our results is that G4227 is only partially methylated, with the unmethylated population allowing the exposure of U4226 . This result is consistent with the data obtained using Ribometh-seq as well as observations of fractional methylation from primer extension experiments . Those were likely false positives owing to a lack of enough total available control sample 3′-base counting reads for DESeq2 statistical analysis. Thus, while promising, this pilot experiment was not good enough for accurate peak calling. In our experience, highly sensitive and accurate site detection is achievable at ∼12 million reads (sequencer output) per sample.Accurate determination of sites of 2′-OMe using RibOxi-seq relies not only on peak calling of oxidized samples, but also on statistical comparison of signals between oxidized and control lanes. An initial pilot experiment using a small quantity of total RNA in combination with Illumina MiSeq sequencing generated ∼2.5 million reads for each sample . Upon examining alignment with 3′-end only reporting, the pattern was strikingly consistent between experiments, with known sites across 18S and 28S rRNAs represented by strong peaks in oxidized samples with corresponding gaps in control samples mapping to the known sites. After single-base differential expression analysis, 36/40 sites in 18S and 54/66 sites in 28S were detected using a filtering strategy similar to that described above. However, there were also more than 30 new sites detected  in 28S and 18S rRNA. Under such conditions, one round of β-elimination is sufficient for accurate site detection even at low sequencing depth. However, the occurrence of methylation is very likely to be far lower in RNAs, such as lncRNAs and mRNAs, and no instances of this modification have been reported so far in human, other than in 5′-cap structures. In order to detect mRNA modifications, not only will higher sequencing coverage be required, but also perhaps multiple β-elimination steps to greatly increase the probability of 3′-end 2′-OMe exposure. Also, additional β-elimination can be used on rRNAs if the amount of input total RNA (∼7.5 µg per sample) described in the standard protocol is impossible to obtain. The required starting material can be divided by 2–4 for each round of β-elimination added. For step-by-step instructions on performing additional β-eliminations, please refer to the alternative steps in the O-methylated oligos as internal controls, comparisons between different sites is not yet possible. On the other hand, comparison between different samples at the same site appears to be feasible.We have demonstrated that RibOxi-seq is highly sensitive and accurate. However, several limitations still exist. Although the number of sequencing reads required is significantly lower than that for Ribometh-seq, the input RNA material required is in general somewhat higher, at the micrograms level, with the possibility of reduction to a sub-microgram level if using additional β-elimination steps. Also, as described above, owing to the nature of ribose methylated bases being resistant to nuclease and alkaline hydrolysis, it is difficult to detect adjacent modified bases if the distal base is fully methylated. Further, exposure of methylated bases relies on extensive and random digestion. tRNAs and other RNAs shorter than 100 bp are difficult to study because the sizes of fragments that would need to be generated might be quite small and difficult to examine. Finally, the method at the current stage cannot be used as a quantitative tool to compare methylation intensity between different sites, since linker ligation efficiencies using T4 RNA ligases 1 and 2 have been described to have sequence biases . Hence, Table top centrifugeProgrammable thermal cyclerHeat blocks/water bathsNanoDrop 2000 UV-Vis Spectrophotometer 2200 TapeStation Qubit 2.0 Fluorometer NextSeq 550 System (Illumina)Seal-Rite 2.0 mL microcentrifuge tube, natural PA-1 [PA1] cell line PureLink RNA Mini Kit PureLink DNase Set TURBO DNA-free Kit Ultra-pure Benzonase 10× Benzonase buffer (store at 4°C)3 M sodium acetate pH = 5.2Ethanol 100%Ethanol 70%UltraPure Phenol:Chloroform:Isoamyl Alcohol  Acid-Phenol:Chloroform, pH 4.5  NucAway Spin Columns RNA Analysis ScreenTape RNA Analysis ScreenTape reagents High sensitivity D1000 DNA ScreenTape High sensitivity D1000 DNA ScreenTape reagents Linear polyacrylamide 10 µg/µL (Mullins Molecular Retrovirology Lab Short protocol)Sodium meta-periodate Sodium periodate oxidation buffer: 4.375 mM sodium borate, 50 mM boric acid, pH = 8.60.2 mL PCR 8-tube FLEX-FREE strip, attached clear flat caps, natural β-elimination buffer: 33.75 mM sodium borate, 50 mM boric acid, pH = 9.5T4 Polynucleotide Kinase SUPERase• In RNase Inhibitor 20 U/μL RNaseOUT Recombinant Ribonuclease Inhibitor T4 RNA Ligase 2, truncated KQ T4 RNA Ligase 1 DMSO 100%SuperScript III First-Strand Synthesis System Sodium hydroxide 1 NEB buffer: 10 mM Tris-Cl, pH 8.5Q5 High-Fidelity 2× Master Mix Agencourt AMPure XP, 450 mL Qubit dsDNA HS Assay Kit NextSeq 500/550 Mid Output v2 Kit (150 cycles) Shrimp alkaline phosphatase 5′-/rApp/CTGTAGGCACCATCAAT/NH2/-3′3′ Preadenylated DNA linker 5′-/Biosg/rArCrArCrGrArCrGrCrUrCrUrUrCrCrGrArUrCrU-35′ RNA linker (50 µM stock)RANATTGATGGTGCCTACAG-3′5′-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTRAN” represents a customizable randomer sequence that can be used to remove PCR duplicates later in the data analysis. Six-base randomers are used in the experiments, but longer is recommended for higher sequencing depth.)(Important: The “Reverse transcription primer (50 µM stock)i5: 5′-aatgatacggcgaccaccgagatctacacBARCODEacactctttccctacacgacgctcttccgatct-3′BARCODEgtgactggagttcagacgtgtgctcttccgatct-3′i7: 5′-caagcagaagacggcatacgagatBARCODE). with accession number GSE96999.Supplemental Material.The library preparation using standard protocol requires around 4–6 d to complete with a moderate work load, factoring out optimization steps. However, the procedure can be stopped whenever an ethanol precipitation is performed and the sample is resuspended into nuclease-free water. Alternatively, samples can also be left precipitating in 100% EtOH under −20°C indefinitely to increase yield. For step-by-step of the protocol, please refer to the details in the detailed protocol in the 1.Extract total RNA using the PureLink RNA Mini Kit with PureLink on-column DNase.2.Optional: Use the Turbo DNase Kit to further remove contaminating DNA.For cells cultured in 10 cm Petri dishes, a PureLink RNA Mini Kit is used in conjunction with the PureLink on-column DNase set to isolate RNA and remove genomic DNA. Steps for the extraction are detailed in the Purelink Kit's protocol. In case any overexpression system is used, additional post-extraction gDNA removal may be necessary due to an increase in DNA molarity. The TURBO DNA-free Kit has proven effective for such conditions after following its “Rigorous DNase treatment” protocol.3.2O.In a 1.5 mL Eppendorf tube, dilute 45 µg of total RNA into 400 µL with nuclease-free H4.Vortex the mixture and spin for a brief second to collect all liquid at the bottom.5.Place the tube into a 90°C heat block for 3 min to denature the RNA and immediately place on ice for at least 1 min.6.Dilute 1 µL stock Benzonase (341 U/µL) into 500 U/mL using 681 µL of 1× Benzonase buffer. (Always dilute fresh prior to using. Do not freeze.)7.Add 45 µL of 10× Benzonase buffer and 5 µL of diluted Benzonase to the diluted RNA . Incubate on ice for 90 min. 8.g at RT for 5 min.Perform phenol–chloroform extraction. Add 450 µL acid phenol:chloroform and vortex for 10 sec. Centrifuge at 20,0009.Transfer about 450 µL of the supernatant into a new set of 2 mL Eppendorf tubes.10.Ethanol precipitate the RNA. Add 50 µL of 3 M sodium acetate (0.1× volume). Mix well and add 1250 µL of 100% ethanol (2.5× volume).11.Mix well and place on ice for >30 min to precipitate RNA.12.g) at 4°C or RT for ∼30 min.Spin at max .16.2O.Resuspend pellet in 100 µL of nuclease-free H17.Use NucAway spin columns following the kit's protocol. 18.Measure RNA concentration using a Nanodrop.19.Optional: Examine fragmented RNA size distribution using TapeStation 2200 and RNA Screen Tapes. Ensure the pattern has a strong peak spanning the region from 25 to 150 bp. Once the Benzonase digestion time is optimized for the laboratory, this step can be omitted.20.Aliquot 1 µg fragmented RNA for each control replicate and store at −80°C until the 3′-end ligation step.21.Ethanol precipitate 4–6 µg fragmented RNA of each oxidation sample replicate (no wash needed).Important: See the “Alternative steps” section.Each sample set should have at least one control and one oxidation sample. We recommend a minimum of three technical replicates for each. After completing fragmentation and the subsequent two steps until just before the oxidation procedure, the RNA loss should be ∼50%. It is recommended to use an initial total RNA amount of 45 µg (can be lowered upon further optimization), which will yield about 28 µg of fragmented RNA (results may vary for each laboratory). The amount of fragmented RNA recommended for oxidation is 4–6 µg, and 1 µg for nonoxidation. Three technical replicates for all samples require about 25 µg total. The amount of starting RNA can be significantly lowered if using the alternative procedure during oxidation and β-elimination steps. Such an alternative is also necessary if applying the method on other samples depleted of rRNAs. Refer to the “Alternative steps” section.4.22.4 solution by dissolving 42.78 mg of the NaIO4 powder in 1 mL oxidation buffer. Protect the solution from light. Important: This step should be performed while precipitating the fragmented RNA and not earlier.Freshly prepare 200 mM NaIO23.4 solution. Mix well and incubate at room temperature protected from light for 45 min (briefly vortex and spin the reaction tube every 10 min to ensure proper resuspension of the pellet).Dissolve RNA pellet in 30 µL oxidation buffer (make sure the pellet is well resuspended) and add 10 µL of prepared NaIO24.2O.Adjust the volume to 90 µL with nuclease-free H25.Perform ethanol precipitation. (Incubate on ice for >30 min or −20°C overnight. No wash needed.) Important: After adding 100% ethanol for precipitation and centrifugation, white precipitates/film might be seen scattered on the Eppendorf tube wall due to high salt content and a typical pellet might not be visible. This is normal. RNA can be recovered if the orientation of the tube is kept consistent and a pipettor is used to vigorously flush and/or gently scrap the precipitates/film off the wall after adding elimination buffer.To prepare the fragmented RNAs for subsequent elimination of the 3′-end bases to expose ribose methylated bases that are potentially positioned one base upstream, these ends need to be oxidized into dialdehydes using NaIOO-methylated bases at the 3′-end of the fragments.26.Add 50 µL of β-elimination buffer to dissolve the pellet. Within 5 min, vigorously vortex, pipette up and down, or invert the tube to further resuspend the oxidized RNA.27.Spin the tubes briefly and transfer samples into PCR strip tubes. Tubes with individual caps are highly recommended to prevent cross-contamination, especially in later steps where control samples are handled alongside oxidation samples.28.Using a thermal cycler, incubate at 45°C for 85 min.29.Recommended: Process samples through NucAway spin columns to remove unwanted small fragments and salts from the samples .30.Transfer samples into a new set of 1.5 mL Eppendorf tubes.31.2O as in the previous oxidation step. Addition of 1 µL LPA is highly recommended to help precipitating and visualizing nucleic acids without disrupting subsequent enzymatic reactions.Ethanol precipitate and resuspend in 22 µL HThis step catalyzes the leaving of the 3′-end oxidized base to expose potentially 2′-O-methylated, another NaIO4 treatment is needed. However, after β-elimination, RNA fragment 3′-ends will contain a mixture of 3′-phosphates, 2′-phosphates, and/or 2′-3′-phosphates. It is vital to remove these phosphate groups to avoid false positives in the final data representation. T4 PNK is used to remove all three types of phosphates.32.Transfer to PCR tubes.33.Remove 3′-, 2′-, and 2′–3′-cyclic phosphate using T4 PNK. (Important: The PNK buffer used must not contain ATP.)34.Incubate at 37°C for at least 4 h .35.Phenol–chloroform extract the dephosphorylated RNA and ethanol precipitate .36.4 solution by dissolving 42.78 mg of the NaIO4 powder in 1 mL oxidation buffer. Protect the solution from light.Freshly prepare 200 mM NaIO37.Dissolve the RNA pellet thoroughly in 30 µL oxidation buffer.38.4 solution. Mix well and incubate at room temperature protected from light for 45 min (briefly vortex and spin reactions every 10 min to further ensure proper resuspension of the pellet).Add 10 µL of prepared NaIO39.2O. If white precipitates remain in the solution, do not remove them.Transfer to a new 1.5 mL Eppendorf tube and ethanol precipitate RNA  and resuspend in 16 µL H40.Following these steps, the samples are properly oxidized.To oxidize all 3′-ends that are not 2′-O-methylated RNA fragments. From this step on, control samples will be subjected to the exact same procedures.41.Thaw the control samples and dilute each into 8 µL. Transfer into PCR tubes.42.Transfer 8 µL of each 16 µL oxidized sample into PCR tubes. Store the remaining 8 µL at −80°C as backup.43.Ligation of 3′ linker .44.Incubate the reaction in thermal cycler at 16°C overnight for 18 h.Ligation of 3′ linkers to unoxidized RNA 3′-ends will enable selective reverse transcription and thus, enrichment of 2′-45.Add 1 µL of the 50 µM RT primer and 69 µL nuclease-free water to each sample.46.90°C for 2 min65°C for 10 min4°C for 1 minIncubate in thermal cycler with the following program:47.2O.Phenol–chloroform extract and ethanol precipitate (add 1 µL of LPA) each sample and resuspend in 11 µL HAny 3′ linker not ligated in the previous step will still be freely available for ligation in the samples. To ensure the 5′ RNA linker ligation attaches the RNA linkers to sample RNA fragments but not to the free 3′ linkers during the next step, the RT primer is annealed first.48.Thaw 50 µM RNA linker from −80°C and transfer (number of samples) *1.3 µL into a PCR tube.49.Denature RNA linker at 72°C for 2 min and return to ice.50.Prepare the following ligation reaction.51.Incubate at 25°C for 1 h, then terminate reaction at 65°C for 15 min.The double-stranded structures resulting from annealing the RT primers and free 3′ linkers will prevent them from being ligated to the 5′ RNA linkers .48.Thaw52.Prepare RT reactions to generate a cDNA library using the following setup with SuperScript III included reagents.53.Incubate the reactions in thermal cycler following the kit's protocol.54.Hydrolyze remaining RNAs by adding 4.4 µL of 1 N NaOH and incubate at 98°C for 20 min.55.Add 22 µL 200 mM Tris-HCl PH = 7.0 to neutralize the PH.56.Use Ampure XP beads at 0.8:1 ratio (add 53 µL Ampure XP solution). Incubate for 5 min to let beads bind cDNA of sizes 250 bp and above.57.Transfer supernatant to new tubes (discard beads) and add an additional 67 µL Ampure XP solution to make the ratio 1:1.8. Finish Ampure XP purification.58.Elute using EB buffer.Because the RT primer has been annealed in the previous step, the RT reaction can proceed without addition of primer. During this step, cDNA is synthesized. At the same time, random-hexamer sequences built into the RT primer are also incorporated into the cDNA library. These will allow the removal of PCR duplicates later during data treatment.Illumina i5 and i7 PCR primer sequences have sequences complementary to the 5′ RNA linker and RT primer sequences. This allows direct PCR amplification of the cDNA library. Periodate oxidation enrichment in previous steps results in a cDNA library of very low complexity. Hence it necessitates additional amplification cycles compared to construction of other types of sequencing libraries (∼35 cycles versus ∼12 cycles). The strandedness of the final library is second-strand similar to the library prepared using the Illumina ligation method.59.Prepare NEB Q5 PCR reactions.60.Incubate in the thermal cycler using the following program modified from the Q5 protocol.61.Add 25 µL of AmpureXP to achieve a 1:1 ratio to select for fragment of sizes ∼200 bp and above, reducing the amount of non-insert fragments .62.Purify libraries following the AmpureXP protocol. Resuspend each sample in 15 µL with Illumina RSB (resuspension buffer).63.Use TapeStation and DNA Screen Tape to visualize library size distributions. It should have a distribution around 200 bp. Even though stringent steps have been taken to avoid non-insert PCR products, they may still be present and are expected.64.Accurately measure library concentrations using the Qbit Fluorometer and dsDNA High Sensitivity Reagent Kit (follow Qbit protocol).65.Calculate library molarity using sizes and concentrations measured.The NextSeq 500 (or MiSeq) 150 cycle Mid Output Kit is used in this experiment with 75 bp by 75 bp configuration.66.Prepare and load the libraries for the NextSeq 500 sequencer following the established protocol. Important: Make sure PhiX phage DNA comprises at least 30% of the total library if the Riboxi-seq samples are the only samples being sequenced because of the nature of extremely low diversity amplicon sequencing. The final loading concentration used in this experiment is 1.5 pM, which is slightly lower than the 1.8 pM from the protocol.67.Remove read-through sequences. Because of the nature of the sequencing library preparation, many fragments will have insert sizes significantly smaller than the 75 bp length sequenced by the sequencer. The resulting read-through sequences will greatly impact alignment reliability. Cutadapt , a Pytho68.TopHat2 is used to align the reads. The following options are supplied in addition to the basic command: -p 12 -r 10 -N 1 - -b2-L 10 - -segment-length 15 - -segment-mismatches 1 - - library-type fr-secondstrand - -no-mixed - -no-discordant -T - -no-coverage-search -G rRNA_annotation.gtf - - no-novel-juncs. The annotation file used consists of chrUn coordinates, which contain two sets of 18S and 28S rRNAs, extracted from the hg19 index .69.The “accepted_hits.bam” for each sample are sorted using SAMtools and converted to “.BED” files using BEDtools  of each read, while the sixth column indicates sense or anti-sense. We counted the number of reads with 3′-end alignment for each position corresponding to 18S and 28S rRNA and generated a count table for each sample .71.The count data files were then imported into DESeq2 for differential analysis per base position .This procedure should be performed between steps 21–22 and between steps 34–35 in the original standard protocol for low starting RNA material or for samples where rRNA has been depleted.1.4 solution. Mix well and incubate at room temperature protected from light for 45 min (briefly vortex and spin the reaction tube every 10 min to ensure proper resuspension of the pellet).Dissolve the RNA pellet in 30 µL oxidation buffer (make sure the pellet is well resuspended) and add 10 µL of prepared NaIO2.2O.Add 10 µL sodium acetate and adjust the volume to 100 µL with nuclease-free H3.Perform ethanol precipitation (no wash needed).4.Add 50 µL of β-elimination buffer to dissolve the pellet. Within 5 min, try as much as possible to vortex, pipetting up and down, or inverting the tube to further resuspend the oxidized RNA.5.Spin the tubes briefly and transfer samples into PCR strip tubes. 6.Using a thermal cycler, incubate at 45°C for 85 min.7.Transfer samples into a new set of 1.5 mL Eppendorf tubes.8.2O as in the previous oxidation step. Dissolve thoroughly.Ethanol precipitate and resuspend in 31 µL H9.Transfer to PCR tubes.10.Prepare the following reaction:11.Incubate at 37°C for 30 min. Stop reaction by heat-inactivation at 65°C for 5 min.12.Phenol–chloroform extraction and ethanol precipitate (add 1 µL LPA) RNA into pellet.13.Repeat alternative steps 1–12 as needed (may require optimization).14.Continue to step 22 in the standard protocol.15.The following steps to phosphorylate fragment 5′-end are required between steps 34 and 35 of the standard protocol because rSAP removes both 5′ and 3′ phosphates.16.2O.Ethanol precipitate reaction and resuspend with 29 µL H17.Prepare the following reaction:18.Incubate at 37°C for 60 min.19.Continue to step 35 in the standard protocol.Supplemental material is available for this article.


E2F3a and caspase 8 associated protein 2 (CASP8AP2) are associated with poor prognosis of childhood acute lymphoblastic leukemia (ALL).Low expression of CASP8AP2 transcription. The direct binding of E2F3a with the promoter of CASP8AP2 was shown by Chromatin Immunoprecipitation (ChIP). Cell proliferation activity and cell cycle were determined by MTS and flow cytometry in leukemic cells after treating with common chemotherapeutic drugs vincristine and daunorubicin.Dual-luciferase reporter assay and wild type as well as four mutated types of reporter plasmids were used to demonstrate the activation of E2F3a on CASP8AP2 promotor (− 206 to − 69 and − 677 to − 507), and activate its transcription activity which was reduced in mutated promotors. The effect of E2F3a on chemotherapeutic sensitivity of leukemic cells could be reversed by down-regulating CASP8AP2.In this study, we found that up-regulation of E2F3a in leukemic cells led to increased fraction of cells in S and G2/M phase, accelerated proliferation, and enhanced sensitivity to vincristine and daunorubicin. ChIP and luciferase assay indicated that E2F3a could directly bind to two fragments in the wild type of CASP8AP2. The effect of E2F3a on chemotherapeutic sensitivity of ALL cells was implemented by regulating CASP8AP2 expression to a great extent.E2F3a could promote transcription and expression of  Leukemia is the most common malignancy in children . HereintCASP8AP2 mRNA was correlated to high level of minimal residual disease (MRD) and relapse [CASP8AP2 in childhood ALL.Caspase 8 associated protein 2 (CASP8AP2) is a multifunctional protein participating in apoptosis mediated by FAS and tumor necrosis factor α , S phase relapse . HoweverE2F3 expressed in most patients, whereas other members’ expression was low or undetectable [CASP8AP2, low expression of E2F3a was common and related to poor treatment outcome in childhood ALL [E2F family was thought of as one of the main transcription regulators of replication dependent histone, and played an important role in regulation of cell cycle progression . Furthertectable , 16. Thetectable , 18. Thetectable . Our prehood ALL .CASP8AP2 transcription directly; its over-expression enhanced the sensitivity of leukemic cells to chemotherapeutic drugs, and could be counteracted by knock down of CASP8AP2. Thus, in childhood ALL, the prognostic significance of E2F3a was largely implemented through regulating CASP8AP2 expression.In the present study, we showed that E2F3a could activate CASP8AP2 nucleotides 416 to 434 (5′-GATTGTCTGAGTTTCCACA-3′). Tet-on recombinant lentivirus for over-expression of E2F3a and negative control (lentivirus encoding red fluorescent protein) were products of Shanghai Genechem Co., Ltd.Recombinant plasmid pcDNA3-E2F3a was a kind gift from Professor Kiyoshi Ohtani at Kansai University, Japan. shCASP8AP2, a tetracycline-inducible short hairpin RNA (Tet-on shRNA) expression plasmid, was kindly given by Professor Vincenzo de Laurenzi of Sapienza University of Rome, which was generated from human CASP8AP2 (GenBank AL353692.14), at − 614, − 169, − 131 respectively. The DNA fragment containing these three binding sites was amplified using 5′-CATATGCCATGGGCCCCTATGACAACTTACCC-3′ as forward primer and 5′-CGAATAAGATCTGGTGGCCTTATTTTCGCACC-3′ as reverse primer. Underlined letters represented the two introduced restriction digestion sites (NcoI in forward primer and BglII in reverse primer respectively). After digested by NcoI and BglII, PCR product was subcloned into vector pGL3-basic to make luciferase reporter CASP8AP2-wild.There were three E2F binding sites (TTTSSCGC) in the 1000 bp sequence upstream of the transcription start site of Based on CASP8AP2-wild, four mutated reporters were generated using Fast Mutagenesis System : CASP8AP2-mutant1, -mutant2, -mutant3, -mutant4, which were mutated at binding site − 169, − 131, − 617, and all the three sites respectively  supplemented with 10% fetal bovine serum (FBS) and penicillin/streptomycin. Human leukemia SUP-B15 cells were cultured in Iscove’s modified Dulbecco’s media (IMDM) supplemented with 20% FBS. Human colorectal cancer HCT116 cells were cultured in DMEM supplemented with 10% FBS. All cells were maintained at 37 °C in a humidified atmosphere containing 5% COTo transfect plasmids, Lipofectamine 2000  and ECM830 Electroporation System  were used for HCT116 and human ALL cell lines respectively.® Reporter Assay System . Relative luciferase activity was represented by FL/RL.Transfected cells were collected after 48 h. Activities of firefly luciferase (FL) and renilla luciferase (RL) were determined using Dual-Luciferase4 per well. After treatment with Doxycycline (0.5 μg/ml), chemotherapeutic drugs , or plasmid transfection, the cells were cultured for 24 h at 37 °C in a humidified atmosphere containing 5% CO2. Twenty microlitre of CellTiter 96 AQueous One Solution Reagent  was added to each well and mixed gently. After incubation for 2–4 h, absorbance at 490 nm was determined using microplate reader. After calibrated with non cellular background, cell viability was calculated using a non treatment control regarded as 100% of cell viability.Cell proliferation was determined by MTS method. Briefly, cells were plated into 96-well plate, 100 μl/well. For HCT116, cell confluence should be about 30%; for leukemic cells, the number of cells should be about 10E2F3a by Doxycycline .697 and REH cells were infected by inducible Tet-on E2F3a lentivirus (multiplicity of infection = 100). Puromycin (0.4 μg/ml) was added after 48 h. Culture medium was changed every two days until the sixth or eighth day, at that time the stable infected cells were screened out. These cells could be induced to over-express E2F3a over-expression by Dox, the 697 cell stably infected with inducible Tet-on E2F3a lentivirus was cultured for 24 h in complete medium containing 10% fetal bovine serum. The harvested cells were washed using phosphate-buffered saline, fixed by 75% ethanol, and digested by RNase A. After nucleus staining by 5 μl of propidium iodide (PI), the cell cycle phase fractions was determined by flow cytometry.Flow cytometry was used to determine the changes in cell cycle phase fractions. Briefly, Dox was firstly tested for its effect on cell cycle phrase fractions in 697 cell. After serum deprivation and induction of CASP8AP2 promotor respectively.The ChIP assay was carried out with ChIP assay kit , anti-E2F3 or rabbit IgG antibody. The separated DNA was amplified with the PCR primer pairs, 5′-GCCCCTCTCTTAAAGCCC-3′ and 5′-GAGTGCCTACGGCCAATCAT-3′ for fragment − 206 to − 69, 5′-GGGTGGAATACCTGACTTGC-3′ and 5′-GCAAGTCAGGTATTCCACC-3′ for fragment − 677 to − 507 in ® Nuclear and Cytoplasmic Extraction Reagents  and NuPAGE Bis–Tris Mini Gels System . Western blotting was carried out with antibodies described below.Total proteins were extracted from cells lysed with RIPA lysis buffer and separated using SDS-PAGE. Nuclear proteins were separated with NE-PERThe antibodies used in this study included anti-E2F3 , rabbit polyclonal anti-CASP8AP2 , rabbit polyclonal anti-β-actin , goat polyclonal anti-Lamin B , Dylight 800 AffiniPure goat anti-rabbit IgG and anti-mouse IgG.E2F3a at different time points. Nonlinear regression curve fit was used to draw the curve and independent-samples T test was used to compare the regression coefficients of the two curves after VCR or DNR treatments in 697 and REH cells with or without over-expression of E2F3a, and in 697 cell over-expressing E2F3a with or without knock-down of CASP8AP2. Relative fluorescence intensities among the groups transfected with different plasmids were compared using one-way Analysis of Variance (ANOVA) or Fisher’s Least Significant Difference (LSD) test. It was regarded as statistically significant when P < 0.05. The statistical analyses were carried out using SPSS16.0 software package. The fitting curves of inhibitory effects of chemotherapeutic drugs on cell proliferation were plotted by GraphPad Prism 5, and half maximal inhibitory concentration (IC50) was also calculated by the software.Independent-samples T test was used to compare cell numbers in 697 cell with or without over-expression of E2F3a was significantly up-regulated by 24 h induction of Dox (0.5 μg/ml) in 697 stably infected with Tet-on lentivirus . By contrast, obvious increases in S and G2/M phases cells were observed  and daunorubicin (DNR) were two main chemotherapeutic drugs in induction therapy of ALL. Different concentrations of VCR and DNR were used to treat leukemic cells for 24 h. In 697 and REH cells, over-expression of E2F3a was induced to over-express  of E2F3a and CASP8AP2 were associated with better treatment response and prognosis; moreover, E2F3a could bind CASP8AP2 promotor and activate its transcription. Thus, the role of CASP8AP2 played in the effects of E2F3a on chemotherapeutic sensitivity was investigated further.As described above, E2F3a could enhance the sensitivity of 697 and REH cells to chemotherapeutic drugs. It was noticed that high expressions of both P = 0.0105). Increase tendency was also found after treatment with DNR . Taken together, the advancing effect of E2F3a on chemotherapeutic sensitivity of ALL cells was implemented by regulating CASP8AP2 expression to a considerable degree, and could be reversed by down-regulating CASP8AP2.shCASP8AP2 led to down-regulation of CASP8AP2 in 697 stably infected with Tet-on E2F3a lentivirus  and poor prognosis [CASP8AP2 decreased gene expression and induced resistance to chemotherapeutic drugs [CASP8AP2 expression. Thus, the key to clarify the mechanism of low expression of CASP8AP2 in childhood ALL was to identify the transcription factors capable of binding core promotor of CASP8AP2.The expression level of CASP8AP2 was an important independent prognostic factor in childhood ALL. The patients with low expression of CASP8AP2 at diagnosis were at greater risk of relapse . Howeverrognosis . It was ic drugs , 23, it CASP8AP2 promotor. The studies of gene expression profiling of St. Jude Children’s Research Hospital and us have indicated that only E2F3 expressed, whereas the expression of other members including E2F1, E2F2, and E2F 4–6 could not be detected in most samples [CASP8AP2 expression in childhood ALL. In present study, E2F3a over-expression could activate CASP8AP2 transcription; mutations of the three E2F binding sites at − 614, − 169, and − 131 made transcription activity decreased significantly. Meanwhile, the binding of E2F3a with the two fragments of CASP8AP2 promotor, − 206 to − 69 and − 677 to − 507, was identified by ChIP (Fig. CASP8AP2 promotor and activate its transcription. It should be noted that as dual luciferase assay showed, relative luciferase activity was still higher in the reporter with three mutations at − 614, − 169, and − 131 than in the control. This was probably due to the existence of other binding sites of some transcription factors, such as c-Myb, c-Myc, and AP-1, which had the potential activating function on the promotor. Thus, further studies were needed to clarify the mechanism of low expression of CASP8AP2 in childhood ALL.There were three binding sites of E2F located at − 614, − 169, and − 131 in  samples , 16. TheCASP8AP2 transcription could coordinate genomic DNA replication and histone biosynthesis, with the result of chromatin integrity and stability. The similar results in colorectal cancer cells and leukemic cells suggested that this regulation probably existed in many types of tissues and cells, and was essential to cell proliferation.E2F3a was the key factor of controlling DNA replication and transition of G1 to S phase . In the E2F3a has been found in many types of solid tumors [E2F3a was generally low-expressed and associated with leukemic relapse or induction failure in childhood ALL [E2F3a over-expression made leukemic cells proliferate more rapidly, and more sensitive to VCR and DNR. It has been long recognized that slow-proliferating leukemic cells were resistant to common chemotherapeutic drugs including VCR, DNR, cytarabine, methotrexate, and thioguanines [CCNB2, CDC2, CKS1B, and interestingly, CASP8AP2 [Abnormal high expression of d tumors , 14, 19,d tumors , 25. Howhood ALL . Accordiguanines . As E2F3It was noteworthy that although E2F3a overexpression reduced IC50 of leukemic cells, there was no significant difference in the viability of leukemic cells at high concentrations of chemotherapeutic drugs. It meant that the effect of E2F3a expression level on chemoresistance was related to drug concentration. High concentration of drugs or combined chemotherapy would play a bigger role in eliminating leukemic cells.CASP8AP2 which was also associated with leukemic relapse in childhood ALL [E2F3a.Although our results were contrary to that of studies in solid tumors; the reason behind this contradiction was unclear. Based on the results of present study, down-regulation of hood ALL  may be oCASP8AP2 was knocked down in 697 with over-expression of E2F3a and sensitivity to VCR and DNR was determined. As expected, IC50 of VCR and DNR treatment significantly increased or tended to increase respectively. Therefore the advancing effects of E2F3a on sensitivity to chemotherapeutic drugs were implemented via up-regulating CASP8AP2 to a great extent. On the other hand, as VCR and DNR were cell cycle specific or non-specific drugs respectively, these results also suggested that the effects of CASP8AP2 on sensitivity to drugs was mainly implemented via influencing cell cycle progression, whereas E2F3a may make effects on chemotherapeutic sensitivity via other ways to be investigated.To test this hypothesis, CASP8AP2 was an important direction for further study.Many mechanisms and factors were involved in resistance to chemotherapeutic drugs of tumor cells, such as DNA damage and repair, cell apoptosis, metabolism and absorption of drugs, and abnormal cell cycle . Except CASP8AP2 promoter directly and activate CASP8AP2 transcription; CASP8AP2 down-regulation reversed the advancing effect of E2F3a on chemosensitivity.Our data suggest that E2F3a increased leukemic cells in S and G2/M phases, accelerated proliferation and enhanced sensitivity to chemotherapeutic drugs. Furthermore, E2F3a could bind


V7.2-KV7.5 channels, has gained much interest for its potential in developing treatments for hyperexcitability-related disorders such as epilepsy. Retigabine, a KV7 channel opener, has proven to be an effective anticonvulsant and has recently also gained attention due to its neuroprotective properties. In the present study, we found that the auxiliary KCNE2 subunit reduced the KV7.2-KV7.3 retigabine sensitivity approximately 5-fold. In addition, using both mammalian expression systems and cultured hippocampal neurons we determined that low μM retigabine concentrations had ‘off-target’ effects on KV2.1 channels which have recently been implicated in apoptosis. Clinical retigabine concentrations (0.3–3 μM) inhibited KV2.1 channel function upon prolonged exposure. The suppression of the KV2.1 conductance was only partially reversible. Our results identified KV2.1 as a new molecular target for retigabine, thus giving a potential explanation for retigabine’s neuroprotective properties.Enhancement of neuronal M-currents, generated through K Epilepsy is a complex, debilitating neurological disorder affecting ~1% of the world’s population. Currently, management of epileptic seizures consists of pharmacotherapy; however, in ~20–30% of patients, seizure control cannot be achieved with conventional treatment strategies134+ (NaV) channels or NMDA/GABA-neurotransmission, retigabine primarily targets voltage-gated K+ channels168V7.2-KV7.5 subunits encoded by KCNQ2–5 genes respectively. Accordingly, many mutations in the KCNQ2 and KCNQ3 genes give rise to distinct epileptic phenotypes further underlining the significance of M-current’s in regulating neuronal excitability1011121314V7 channels by interfering with the normal gating behavior, i.e. retigabine shifts the voltage-dependence of activation to hyperpolarized potentialsV7-mediated currents activate at more negative membrane potentials, effectively hyperpolarizing the resting membrane potential. The binding of retigabine to KV7 channels occurs near the pore domain and is dependent on a conserved Trp residue (KV7.2 Trp236) that is absent in the KV7.1 channel primarily expressed in cardiac and epithelial cells17Retigabine (RTG) is a novel, ‘first-in-class’ anticonvulsant drug approved for use in partial-onset seizures6V7 activators interesting compounds for several other hyperexcitability-related disorders such as migraine, chronic pain, tinnitus, and even Huntington’s disease2021222425V7 channels since it also modulates GABAA receptors in a similar concentration range2829V7 channels remains unknown. Surprisingly, despite the large structural similarities within the KV channel family, little effort has been made to determine whether other KV channels are modulated by retigabineV7 despite the fact that KV7.2-KV7.3 channels and KCNE2 have overlapping expression patterns and described gating effects31This general mechanism for suppression of neuronal excitability additionally makes retigabine and other KV1–KV9 and KV11 subfamilies to investigate whether these channels are affected by retigabine. We found that retigabine inhibited all KV channels tested, but that this inhibition only occurred in the high μM range, with the exception of KV2.1. Inhibition of KV2.1 required only low μM concentrations and was only partially reversible. In addition, we found that the addition of the auxiliary subunit KCNE2 decreased the retigabine sensitivity of heterotetrameric KV7.2-KV7.3, but not of KV2.1, channels. These findings identify KV2.1 as an important molecular target for the action of retigabine and, due to KV2.1’s key role in apoptosis, could help explain the previously reported neuroprotective properties2425Here, we performed an electrophysiological screening on members of the KV7 channels is highly dependent on its association with auxiliary KCNE subunits33V7.2-KV7.3 channels - based on overlapping expression patterns and gating effects31V7.2-KV7.3 with YFP-KCNE2 enabling us to select KCNE2-transfected cells. Retigabine potentiated KV7.2-KV7.3 currents in a concentration-dependent manner  was reduced from 39.2 ± 1.4 mV (n = 11) under control conditions to 30.7 ± 1.1 mV (n = 9) in the KCNE2-transfected cells . However, KV2.1 currents were inhibited at relatively low concentrations (10 μM)  . InteresV2.1 channel inhibition, we performed a detailed biophysical characterization of retigabine effects. Retigabine inhibited KV2.1 currents with an IC50 of 22.0 ± 1.6 μM and Hill coefficient of 1.6 ± 0.1  and a Hill coefficient of 1.4 ± 0.2 (p = 0.384) (n = 6). No change could be observed in the voltage-dependence of inactivation  of the current at +60 mV while only 27.0 ± 12.4% (n = 7) inhibition occurred at −10 mV , on average requiring ± 10 minutes before inhibition was saturated (wash-out of 574 ± 67 s (n = 8), and incomplete with only 41.8 ± 7.6% recovery in 30 minutes  did not substantially inhibit other KV channels , a selective Kv2 inhibitorV2-mediated currents in mice CA1 hippocampal neuronsV2-mediated currents. To validate these results, we performed the experiments in the reverse order: KV2-mediated currents were first inhibited with GxTx-1E, before retigabine was applied  effects. Although acute exposure of KV2.1 channels to retigabine resulted in inhibition at concentrations above the clinical range endocrine cells43V2.1 channels constitute the major delayed rectifier current in hippocampal neurons, and targeted deletion of KV2.1 results in neuronal and behavioral hyperexcitability3745V2.1 channels is intriguing, in that they form cell-surface clusters at the soma, proximal dendrites and the axon initial segment, not only in cultured hippocampal neurons and intact brain but also in transfected HEK cells47V2.1 channels are inactive, i.e. gating charge movement of the voltage sensing domains was detected without measurable ionic currents, and upon dispersal, the ionic current is regained. Within the micro-domain of the cluster, KV2.1 channels are in close proximity to the endoplasmatic reticulum (ER) and induce the formation of ER-plasmamembrane junctions4950V2.1 cell surface clusters are insertion platforms for ER-membrane traffickingV2.1 is highly dependent on the phosphorylation state of the channel and directly linked to underlying neuronal activity. This means that hyperexcitability, as observed during epileptic seizures, promotes dephosphorylation of KV2.1 through a Ca2+/calcineurin-dependent mechanism which leads to increased KV2.1 activity and KV2.1 declustering3653V2.1 clusters can also be dispersed pharmacologically. When hippocampal neurons are exposed to glutamate, rapid KV2.1 declustering occursV2.1 currents in a poorly reversible manner, both in HEK cells and hippocampal neurons, KV2.1 trafficking might be affected upon retigabine exposure. One could speculate that loss of functional KV2.1 channels at the cell surface, due to increased endocytosis or changed clustering pattern, might contribute to retigabine’s poorly reversible inhibition of Kv2.1 currents. However, using live-cell imaging of GFP-tagged KV2.1, we did not observe a change in the KV2.1 localization within 30 min after exposure to retigabine (data not shown). Interestingly, KV2.1 current densities were reduced with much lower retigabine concentrations (0.1–3 μM) upon prolonged exposure. In this case, the reduced KV2.1 current density might simply reflect the population of KV2.1 channels that were not inhibited by retigabine as the gating and trafficking (data not shown) properties were not modified. In addition, it cannot be excluded that the observed reduction in KV2.1 current density is  caused by a drug metabolite of retigabine. On the other hand, retigabine’s hydrophobic nature suggests that it might reside in the lipid bilayer or bind to a hydrophobic region in the KV2.1 channel. Although this could help explain the poor reversibility of KV2.1 current inhibition upon wash-out, it seems rather unlikely, because: 1) increased solvent concentrations had no significant effect on the reversibility of inhibition  V2.1 might be involved in the neuroprotective properties of retigabine that have been described more recently, especially due to the key role of KV2.1 in apoptosis56V2.1-currents are increased through de novo insertion of channels in the plasma membrane and subsequent decrease of intracellular [K+] levels promoting activation of the apoptotic cascadeV2.1 current is pharmacologically inhibitedV2.1 currents, thus promoting cell survival. However, retigabine was found to promote neuroprotection by diminishing excitotoxicity through suppression of hyperexcitability by KV7 channel activation2425V2 and KV7 channels.Interestingly, KV7 channels is reduced by the auxiliary KCNE2 subunit. In addition, retigabine inhibited KV2.1 channels most likely through an open-channel block mechanism in a poorly reversible manner at clinically relevant concentrations.In conclusion, we found that the retigabine sensitivity of KV1.5 (GenBank Accession Number NM_002234), KV2.1 (NM_004975), KV3.1 (NM_004976), KV4.2 (NM_012281), KV5.1 (NM_002236), KV6.4 (NM_172347), KV8.1 (NM_014379) and KV11.1 (NM_000238), as well as mouse KV9.3 (NM_173417), were subcloned in the eGFP-N1 vector . Human KV7.1 (NM_000218), KV7.2 (NM_172107), KV7.3 (NM_004519), KCNE2 (NM_172201) and YFP-KCNE2 were subcloned in the pBK/CMV vector as described previouslyHuman K2). HEK293 cells were transiently transfected with 0.05–5 μg cDNA of the respective channel together with the GFP transfection marker using Lipofectamine2000 , according to the manufacturer’s instructions. To obtain the characteristic KV7.2-KV7.3 currents both KV subunits were co-transfected in a 1:1 molar ratio. Co-transfection with KCNE2 was performed in 1:1:4 and 1:4 molar ratios for KV7.2-KV7.3 and KV2.1 channels, respectively. The amount of cDNA was kept identical between the–KCNE2 and +KCNE2 experiments by addition of empty vector cDNA. A C-terminal YFP-KCNE2 construct was used to allow for selection of KCNE2-transfected cells. The YFP-KCNE2 behaved similarly to the untagged KCNE2. 16–24 h after transfection, HEK293 cells were dissociated with trypsine and transferred to the patch-clamp set-up for electrophysiological analysis.HEK293 cells were cultured in 60 mm cell culture dishes filled with 4 ml culture medium - consisting of Dulbecco’s modified Eagle’s medium supplemented with 10% horse serum, 1% penicillin/streptomycin and 1% non-essential amino acids–under physiological conditions  containing 0.1% DMSO (vehicle control) or 0.1, 0.3, 1 or 3 μM retigabine for 4 h. After the 4 h exposure, the incubation medium was removed and fresh medium was added to the transfected HEK293 cells. Thus, at the moment of electrophysiological analysis (48 h post-transfection), no retigabine was present in the recording solution.The retigabine incubation experiments were performed on HEK293 cells transfected with 10 ng Kin vitro and afterwards analyzed with the patch–clamp technique.All use of animals was approved by the institutional animal care and use committee of the University of Copenhagen. All experiments were performed in accordance with the relevant guidelines and regulations. Primary cultures of rat hippocampal neurons were obtained as described previously2, 1 CaCl2, 10 HEPES and 10 Glucose, adjusted to a pH of 7.35 with NaOH. Patch pipettes (1.5–2.5 MΩ) were pulled from borosilicate glass capillaries, heat polished and filled with an intracellular solution (ICS) containing (in mM): 110 KCl, 5 K2ATP, 5 K4BAPTA, 2 MgCl2 and 10 HEPES, pH adjusted to 7.2 with KOH. ICS and ECS solutions were used to record in HEK293 cells as well as in native hippocampal neurons. Where mentioned, 5 μM TTX was added to the ECS solution to inhibit native Na+ currents. When leak currents exceeded 10% of the total ionic current or voltage errors at the highest used potential exceeded the cut-off value of 5 mV (after series resistance compensation), cells were excluded from analysis.Whole-cell ionic currents were recorded as previously describedAll drug solutions were applied to the HEK293 cells using a fast perfusion system . Control recordings were obtained with fast perfusion of ECS solution. Retigabine  was dissolved in DMSO to obtain a stock solution of 100 mM. The working concentrations (0.1–300 μM) were obtained by diluting the stock solution in ECS solution. Guangxitoxin–1E (Alomone Labs) and TTX  were dissolved and diluted in ECS solution to obtain the stock (100 μM and 5 mM) and working (100 nM and 5 μM) solutions.50/[D])nH), was fitted to concentration-effect curves to obtain a relative measurement of drug affinity with EC50 the concentration that induces 50% of the effect and nH the Hill coefficient. For Kv7 channels the effect is defined as the induced shift in the voltage–dependence of activation normalized to the maximal shift (i.e. ΔV/ΔVmax) while for Kv2.1 the degree of current inhibition (%) was plotted. The Boltzmann equation: y = 1/(1 + exp (−(V − V1/2)/k)), was applied to fit the voltage–dependence of (in)activation, where V represents the applied potential, V1/2 the voltage where 50% of the channels are (in)activated, and k the slope factor. Results were reported as the mean value ± S.E.M. Standard t–test or the Mann–Whitney Rank Sum test were used to determine whether the results achieved statistical significance. Statistical significance was defined as P < 0.05. Pulse protocols were adjusted to match the biophysical properties of the respective Kv channel and are illustrated throughout the figures.The Hill equation: 1 − y = 1/.


The synthetic CH3NH3PbI3 nanofibers were characterized by X-ray diffraction, scanning electron microscopy, thermogravimetric analysis, and photoluminescence. As counter electrodes, the synthesized nanofibers increased the performance of the dye-sensitized solar cells from 1.58 to 2.09%. This improvement was attributed to the enhanced smoothness and efficiency of the electron transport path. Thus, CH3NH3PbI3 perovskites nanofibers are potential alternative to platinum counter electrodes in dye-sensitized solar cells.CH Among these materials, semiconducting CHttention . These nttention –3. Spin-reported –6. Notabreported , 7.Currently, dye-sensitized solar cells (DSSCs) have been extensively studied because of their availability and low cost . In DSSC2, ZnO, and Cu2ZnSnS4 , can be used in solar cells [3NH3PbI3 perovskites are similar to those of CZTSs. Thus, we explore the use of CH3NH3PbI3 perovskite nanofibers as counter electrodes in DSSCs.One-dimensional (1D) nanofibers have attracted considerable attention because of their large length-to-diameter ratio, high surface area, excellent aspect ratio, and effective electronic properties . Throughar cells –14. More3NH3PbI3 perovskite nanofiber using an electrospinning technique. We used polyvinylpyrrolidone (PVP) as an electrospinning medium. Subsequently, to explore the application of the synthesized nanofibers, we attempted to use them as a replacement for Pt in DSSCs.As far as we know, we are the first to fabricate CH3NH3PbI3 precursor was prepared by adding CH3NH3I3 and PbI2 at a ratio of 1:1 to 2 mL N,N-dimethylformamide (DMF) (J&K Scientific Ltd.). Approximately 0.25 g of PVP  was then dissolved into the solution. The resulting solution was stirred for 30 min until a homogeneous mixture was obtained. Facile electrospinning technique method was used to fabricate the PVP-CH3NH3PbI3 perovskite nanofibers. The experimental procedure is schematically illustrated in Fig. 3NH3PbI3 nanofibers obtained were calcined at 150 and 200 °C separately in a nitrogen atmosphere for 5 min.CH2 F20, USA). Thermal analyses were performed through thermogravimetric analysis  in a nitrogenous atmosphere. The band gap was measured through the photoluminescence (PL) spectrum.The phase structures of the prepared samples were characterized using an X-ray diffractometer . Field-effect scanning electron microscope (Hitachi S-520) and transmission electron microscope were then used to study the microstructure of the samples , (220), (310), (224), and (314) crystal planes of the tetragonal perovskite structure. This result is consistent with the previously published results [Figure 3NH3PbI3 perovskite nanofibers at different annealing temperatures. Figure 3NH3PbI3 perovskite nanofibers, which are covered throughout the surface. Moreover, the surfaces of CH3NH3PbI3 nanofibers are quite smooth and their diameters ranges from 140 to 170 nm. Figure 3NH3PbI3 nanofibers. The appearance of multiple diffraction rings is due to the random orientation of the polycrystallites. The spotty ring pattern with missing periodicity is due to the random orientation of the particles [3NH3PbI3 nanofibers after annealing treatment at 150 °C are shown in Fig. 3NH3PbI3 perovskite.Figure articles . The res2 atmosphere. The curve indicates that the thermal decomposition for the as-spun nanofibers is completed in two distinct steps. In the first step, the weight loss (3%) is observed between 25 and 200 °C. The loss is due to the evaporation of water and alcohol. In the second step, (200–400 °C), the weight loss is approximately 35% and is due to PVP degradation, which involves intra- and intermolecular transfer reaction mechanisms.The TGA curves are shown in Fig. 3NH3PbI3 nanofiber at different annealing temperatures is shown in Fig. 3NH3PbI3 layer is comparable to the thickness of the layer [The band gap of the semiconductor plays an essential role in counter electrode of the DSSCs . The PL he layer .Fig. 5PL3NH3PbI3 nanofiber has been applied as a counter electrode in DSSCs. The DSSCs are each equipped with a TiO2/FTO working electrode, redox couple (I−/I3−), and CH3NH3PbI3/FTO counter electrodes [3NH3PbI3/FTO counter electrodes. Figure VOC), short circuit current density (JSC), and filled factor (FF) of the device are 0.45 V, 7.78 mA/cm2, and 45%, respectively. When the counter electrodes have been changed from Pt/FTO to CH3NH3PbI3/FTO, the JSC and FF values have increased slightly by 9.81 mA/cm2 and 51%, respectively. Moreover, the conversion efficiency of the device has increased from 1.58 to 2.09% because of the improved efficiency of the electron transport path in the CH3NH3PbI3/FTO electrodes. Meanwhile, the nanofibers with large surface areas contribute to the redox reaction between the counter electrode and the electrolyte, and thus, a decreased interfacial recombination in the DSSCs is observed [The CHectrodes . The curobserved .Fig. 6J3NH3PbI3 nanofibers with diameters ranging from 140 to 170 nm via electrospinning technique. The XRD analysis results revealed that the synthetic nanofibers contained the pure phase of CH3NH3PbI3 perovskites with good crystallinity. The PL properties demonstrated that the nanofibers have a band gap energy of approximately 1.6 eV. When the nanofibers were used to the counter electrodes of the DSSCs, the conversion efficiency of the device increased from 1.58 to 2.09% because of the large surface area of the small nanofibers. Thus, our synthetic method can significantly contribute to low-cost and large-scale preparation of nanofibers for actual photovoltaic applications.In summary, we successfully synthesized CH


Tenofovir (TDF) based regimen was reported to have better immunological outcomes. Unfortunately, there is limited information regarding the immunologic outcome associated with this regimen in Ethiopia, as its routine utilization in this setting begun since 2013.P < 0.05 for slopes of the random effect linear regression was used as indicators for presence of association.A 2 years retrospective cohort study was conducted at Jimma University Specialized Hospital, 346 km Southwest of Addis Ababa, Ethiopia. A total of 280 patients’ data from September 2012 to July 2014 was extracted from records from February 10, 2015 to March 10, 2015. Records were selected using a simple random sampling technique. Data on socio-demographic, clinical and drug related variables were collected; entered into EpiData 3.1 and analyzed by STATA 13.1. Mixed effect linear regression was performed to assess difference in CD4+ change between groups adjusting for baseline characteristics. The change in predicted CD4 count attributed to each regimen was also assessed by marginal analysis. P = 0.753) for TDF and AZT groups respectively. The minimum follow up duration was 7.4 and 8.9 months for TDF and AZT groups respectively. Most of TDF (93.6%) and AZT (91.4%) groups completed their follow up, 5 (3.6%) TDF and 6 (4.3%) AZT groups died and 4 (2.9%) TDF and 6 (4.3%) AZT groups were lost for follow-up (P = 0.769). There was statistically significant difference in immunologic recovery between the groups  over time. The predicted CD4+ count for TDF/3TC/EFV was  whereas that of AZT/3TC/EFV was .The mean (SD) duration of cohort follow up was 714.2 (69.6) and 708.8 (78.9) days  in this set-up among TDF users. Around 1980s, Acquired immunodeficiency syndrome (AIDS) was globally emerged as a major public health threat. The reaction against it has led to unprecedented attention and commitment from the international community to improve access to human immune virus(HIV) care, antiretroviral treatment (ART) and prevention . The intCurrently, there are more than 20 ARV compounds approved for use in United States (US) and Europe. Multiple adult HIV treatment guidelines recommend the nucleos(t)ide reverse transcriptase inhibitors; AZT or TDF based regimens and most are in favour of TDF based regimens as the safer regimen for patients with no contraindication due to its proven effectiveness, favourable toxicity profile, and demonstrated regimen durability , 7–9.Safe and efficacious ART regimens improve patient care through rapidly restoring the immune cells, promoting adherence and alleviating the hazards of mortality , 10, 11.Studies from developed regions showed that there were 18.0 and 18.8% immunologic failures in the EFV/FTC/TDF and EFV/3TC/AZT arms, respectively. There were no significant differences in the risk of HIV-1 disease progression or death . In otheThe study was conducted at Jimma University specialized Hospital which is located in Jimma town; Jimma Zone, 346 km Southwest of Addis Ababa, Ethiopia. The hospital has separate ART clinic with about 7486 clients. The ART clinic provides HIV and TB treatment and care. Patient data from September 2012 to July 2014 was extracted from records from February 10, 2015 to March 10, 2015.A retrospective hospital based cohort study was conducted on adult patients who were on TDF and AZT based regimens and fulfilled inclusion criteria. The study was conducted by dividing the total sample into two groups: TDF and AZT groups.We included patients who were older than 14 years, have at least 6 months follow-up, whose records were legible and complete, and who have at least baseline and sixth month CD4 count. We excluded pregnant women from the study because there is significant pharmacokinetic change that is enough to impact the treatment outcome in this group of population. Those whose regimen was changed were also excluded from the study.Sample size determination was guided by the number of patients on TDF/3TC/NVP where only 70 patients fulfilled the inclusion criteria. Patients from other regimens were selected based on the size of this group to satisfy 1:1 ratio of AZT to TDF group. Therefore, frequency matching was used to select a total of 280 subjects (140 TDF group and 140 AZT group). Records were selected by simple random sampling technique using computer generated random numbers.Data on demographic, clinical, laboratory, drug administered, comorbidities and adherence were collected by record review using English version checklist which was prepared after reviewing different relevant literatures. Baseline body mass-index of the subjects was calculated after collection of baseline height and weight of the patient from patients chart. Data from antiretroviral drugs sheet and patient information sheet were collected by pharmacists, while nurses collected data from ART intake and follow up forms.Data were double entered into Epi-Data 3.1 and exported to STATA 13.1 for cleaning and analysis. Descriptive analyses were performed and results were presented by text, tables and charts. Bi-variate and multivariable mixed effect linear regression for repeated measurements were performed to assess adjusted effect of the ART regimen and identify additional predictors of CD4 recovery. Coefficient of mean CD4 count with 95% confidence intervals was used as measure of strength of association and P < 0.05 was considered to declare a statistical significance. Marginal analysis was also conducted to see the difference among specific regimen category.A total of 1034 patients were on antiretroviral therapy (ART) for at least 6 months during the study period. Nine hundred eighty six had complete CD4+ count at 6 month of treatment. Fourteen records were used for pre-test, 22 and 110 records were excluded because of pregnancy and regimen change respectively. Only 70 patients remained on TDF/3TC/NVP and this governed the sample selection  for TDF and AZT groups respectively. The minimum follow up duration was 7.4 and 8.9 months for TDF and AZT groups respectively. Most of TDF (93.6%) and AZT (91.4%) groups completed their follow up, 5 (3.6%) TDF and 6 (4.3%) AZT groups died and 4 (2.9%) TDF and 6 (4.3%) AZT groups were lost for follow-up (P = 0.769).The cohort was followed for 2 years. The overall time the cohort was at risk was 539.39 years. The cohort contributed to a total of 2.74/100 and 2.72/100 person-years of follow-up for TDF and AZT groups respectively. The mean (SD) duration of follow up was 714.2 (69.6) and 708.8 (78.9) days (2 with mean (SD) of 19.7 (3.4) and 20.4 (3.0) respectively. Larger proportion of TDF (48.6%) and AZT (41.5%) groups were employed. Majority of TDF (69.3% and AZT (78.5%) groups were urban. Concerning alcohol use, most of TDF (80.7%) and AZT (72.9%) groups had history of alcohol use. Regarding between groups comparison of distribution of baseline socio-demographic and behavioural characteristics of patients, there was statistically significant difference between groups only in terms of religion (Table Majority of TDF (64.3%) and AZT (66.4%) groups were females. Majority of TDF 77.1%) and AZT (85.9%) groups were in the age group 25–45 years with mean (SD) of 32.3 (7.4) and 32.3 (9.2) years respectively. Majority of TDF (62.9%) and AZT (73.6%) groups had BMI ≥ 18 kg/m% and AZT3 with mean (SD) of 164.64 (83.36) and 175.21 (89.14) respectively. There was statistically significant difference between groups in terms of baseline CD4 count. Leading percentage (33.6%) both TDF and AZT groups were in WHO clinical stage III and II respectively. Most of TDF (78.6%) and majority of AZT (56.4%) groups had working functional status and there was statistically significant difference between groups. Twenty (15.3%) and 17 (12.1%) of patients were treated for TB from TDF and AZT groups respectively. CPT was prescribed for most of (87.9%) and nearly all of (92.14%) patients in TDF and AZT groups respectively, whereas INH was prescribed for only 26.4 and 21.5% of patients in TDF and AZT groups respectively (Table Majority of TDF (65.7%) and AZT (53.9%) groups had baseline CD4+ count <200 cells/mmThe mean change in CD4+ over study period is shown by Fig. At 6 month, the overall proportion of sub-optimal CD4+ response (net CD4+ gain <50 cells/µl) was 23.95% . Earlier at the initiation of ART, the CD4+ count showed a linear trend but it becomes more flat after 18th month with a very minimal gain irrespective of the regimen.Considering consecutive CD4+ count as an outcome variable, multilevel mixed effect linear regression was performed Table . The slo3  with the conditional correlation coefficient of 64.9%, i.e. 64.9% of the variability in CD4+ count between two visits was explained by unobserved patient specific factors. Among these, 38.99% of the variation in CD4+ change was explained by differences in the regimens where as 25.34% of the variation is attributed to other regression variables (P < 0.001), showing an important heterogeneity between patient groups.Accordingly, the average gain in CD4+ count achieved at every visit among the cohort was 38 cells/mm, P = 0.032).In the overall analysis, keeping other factors constant, older age was found to be one of the negative predictors of CD4+ count change. So younger patients (age <25 years) were found to have +66 gain in CD4+ count every 6 months as compared to those aged ≥45 years (B = −66.19 95% CI 2 had a significantly higher CD4+ change (+32 cell/mm3) over time than those who had BMI < 18.5 kg/m2 .Among the predictor variables, female sex was strongly associated with progressive CD4+ count gain at each visit. So, females had +39 CD4+ cells advantage over time  as compared to males. Patients with BMI ≥ 18.5 kg/mBaseline CD4+ count was also another independent predictor for CD4+ advantage over time . More importantly, patients in TDF group had a significant CD4+ count gain per visit compared with their AZT counterparts .In this study, other baseline patient factors such as WHO clinical stage, TB treatment, educational status, place of residence, religion, occupational status, marital status, alcoholic use and prophylaxis had no significant association with CD4+ change.To predict the expected change in CD4+ count in both groups at the end of treatment period, post-estimation marginal analysis was conducted.3 for patients treated with TDF/3TC/EFV and TDF/3TC/NVP respectively. In AZT group, the predicted mean CD4+ count changes were 319.11 and 281.54 cells/mm3 for patients treated with AZT/3TC/NVP and AZT/3TC/EFV respectively. These figures were exactly the expected increase in CD4+ count associated with each regimen and has a crucial clinical implication in guiding clinicians to choose which regimen to initiate on as the role of good immunologic recovery in treatment of HIV infection is multidimensional. So, this section of analysis . At first 6 months, majority of sub-optimal immunologic responders belonged to TDF/3TC/EFV (30%) and lowest proportion of sub-optimal immunologic responder was observed among AZT/3TC/EFV (17.9%).3) followed by AZT/3TC/EFV (237 cells/mm3) at 96 weeks. Another finding from Nigeria indicated that TDF/3TC/NVP is much more inferior (208 cells/mm3) to AZT/3TC/NVP (221.1 cells/mm3) at 12 months of therapy  in whiB = 0.879 95% CI , P < 0.001). This finding is consistent form study by Lawrence et al. , P = 0.001). This study is consistent with most of the previous findings that described TDF based regimens with better immunologic outcome [From this study, patients randomized to TDF group had a significant CD4+ count advantage per visit relative to patients randomized to AZT group ( outcome , 31, 32.3 and patients with pre-treatment BMI < 18.5 were poor immunologic responders, they need special attention while delivering care and treatment. However, the prevalence of sub-immunologic recovery among the TDF users in the resource constrained settings needs to be assessed further.The results of this study suggest that TDF based regimens especially, TDF/3TC/EFV had excellent immunologic recovery followed by AZT based NVP. Since aged patients, those with baseline CD4+ count <200 cells/mm


Carica papaya cv. Maradol. We isolated four genes , and an in silico analysis showed that the four genes encode proteins that contain a conserved APETALA2 (AP2) domain located within group I and II transcription factors of the AP2/ERF superfamily. Semiquantitative PCR experiments indicated that each CpRap2 gene is differentially expressed under stress conditions, such as extreme temperatures. Moreover, genetic transformants of tobacco plants overexpressing CpRap2.4a and CpRap2.4b genes show a high level of tolerance to cold and heat stress compared to non-transformed plants. Confocal microscopy analysis of tobacco transgenic plants showed that CpRAP2.4a and CpRAP2.4b proteins were mainly localized to the nuclei of cells from the leaves and roots and also in the sieve elements. Moreover, the movement of CpRap2.4a RNA in tobacco grafting was analyzed. Our results indicate that CpRap2.4a and CpRap2.4b RNA in the papaya tree have a functional role in the response to stress conditions such as exposure to extreme temperatures via direct translation outside the parental RNA cell.Plants respond to stress through metabolic and morphological changes that increase their ability to survive and grow. To this end, several transcription factor families are responsible for transmitting the signals that are required for these changes. Here, we studied the transcription factor superfamily AP2/ERF, particularly, RAP2.4 from  The AP2/ERF TF superfamily hosts a highly conserved AP2 domain, which consists of approximately 60 to 70 amino acids and is involved in DNA binding , and incubated for 1 h at 70°C. The solution was then twice extracted with equal volumes of chloroform:isoamyl alcohol  and centrifuged at 15,000 × g, 4°C for 10 min. The aqueous phase was transferred to a 1.5 ml tube, and the DNA was precipitated with an equal volume of isopropanol, and kept at -80°C for 1 h. The DNA was then spooled out and washed with 80% ethanol-15-mM ammonium acetate, and a second wash was performed with 100% ethanol. Each washing step was performed for 20 min with gentle shaking. The DNA pellet was air dried and dissolved in 100 μl of Tris-EDTA buffer  by incubating at 65°C for 1 h.Total RNA from different plant tissues was isolated using the TRIzol Reagent , according to the manufacturer’s instructions. Each cDNA was diluted 1:5, and 2 μl of diluted sample was used in a 30 cycle PCR program with gene-specific primers as follows: The primers used: CpRap2.4a (forward) 5´- ATG CCT CAA CCT ATT TCA AAC GC-3´ and (reverse) 5´- TCA TGA CAA TAT GGA GGC CCA AT-3´); CpRap2.4b (forward) 5´- ATG GCA ACA GCT ATA GAT-3´ and (reverse) 5´- TTA ATA TTC GGA TAG TTT CCT TAT-3´); CpRap2.1 (forward) 5´- ATG GAA GGA GAG TGT TGT TCG ACG-3´ and (reverse) 5´- TCA GTC TTC ATC GGA AGT TTC CGG-3´; and CpRap2.10 (forward) 5´- ATG GAG GGT GGA GCG GAG-3´ and (reverse) 5´- TCA CTC GCC ATC CGA ACT ATC TG-3´). The housekeeping 18S gene was amplified from each sample to normalize the level of each test gene using 18S universal primers . PCR products were run on a 2% agarose gel with ethidium bromide and visualized on a Molecular Imager® Gel Doc™ XR+ System.For semi-quantitative RT-PCR, 1 μg of total RNA was used for the cDNA synthesis. Reverse transcription was performed by SMARTerTM PCR cDNA Synthesis Kit  and then cloned into the pGem-T easy vector system  according to the manufacturer's instructions. The ligations were transformed into E. coli XL1-Blue competent cells using a calcium protocol transformation  methionine at 10 mCi/ml, 0.5 μg/ul of fibrillarin RNA and 25 μl of C. papaya sap in a total volume of 50 μl with nuclease-free water. The sap was suspended in storage buffer . The reaction was incubated at 30°C for 2 hours. The sample was heated at 100°C for two minutes to denature the proteins and then loaded in a 10% SDS-PAGE gel. The gel was allowed to dry at 80°C for 90 minutes in a vacuum chamber and then was subjected to autoradiography. B) Purification of 6His-FIBRILLARIN after in vitro translation. Reaction was loaded on 50 ul Ni-Agarose resin washed and eluted with 250 mM Imidazole. Input (I), flow through (FT), washes (w1-w4) and elution’s (E1-E2) were loaded in the indicated lanes on a 12% PAGE, and silver stain for protein detection. (PDF)The RNA was synthesized in vitro from the pET15b::(PDF)Click here for additional data file.S4 FigCpRap2.4a::gfp mRNA and then incubated for 3 days at 25°C in in MS medium containing glutamine at 0.4-mg/1 10-mg/1 2,4-D, 6% sucrose and 8-g/1 agar with the pH adjusted to 5.8. The embryos were mounted on slides with moviol with DAPI stain and analyzed the GFP fluorescence . 2% of the cells showed nuclear GFP fluorescence from the embryonic tissue that was analyzed using a confocal laser-scanning microscope FV100 Olympus. DAPI staining was used to determine the location of the nuclei in cells. The experiment was carried out in triplicate (100 counted cells each time) and done on two independent times. B) Control callus without RNA incubation.A) The embryogenic callus were wound 5 times with the tip of a insulin syringe that was used to add either the presence of 5 μg (PDF)Click here for additional data file.S5 FigA) MS medium without ABA, B) MS medium supplemented with ABA 1 mM. (PDF)(PDF)Click here for additional data file.S6 FigCpRap2.1, CpRap2.10, CpRap2.4a and CpRap2.4b gene) in their genome. The fluorescence of GFP and DAPI staining were taken at 40X on an Olympus FV1000 confocal microscope. Wild-type Tobacco plants were used as a control. A) leaf and B) root, tissues from CpRAP2.1 and CpRAP2.10 stained with DAPI. C) leaf and D) root, tissues from CpRAP2.4a and CpRAP2.4b. In S6D) merge from GFP and DAPI signals are merge to shows nuclear colocalization. (PDF)The leaves or roots of tobacco seedlings after 8 days of germination were taken under different light sources Transmitted light is observed in the horizontal first line, DAPI staining is observed in the second horizontal line and GFP is observed in the third horizontal line. DAPI staining or GFP fluorescence of leaf or root is shown for tobacco transformed plants that they carry different CpRap genes (the (PDF)Click here for additional data file.S1 FileCarica papaya, and Uptake and translation of CpRap2.4a::gfp in embryogenic callus. (PDF)Methods for RNA expression of CpRap2.4a::gfp, micropropagation of embryogenic culture of (PDF)Click here for additional data file.S1 TableC. papaya tree and that were named CpERFs. The groups were clustered according to Nakano et al. (2006). (PDF)We identified and retrieved 80 sequences with the AP2/ERF domain from (PDF)Click here for additional data file.S2 TableEach band was quantified and normalizes by dividing the value from the 0 time and then multiplying the value by 100. (PDF)(PDF)Click here for additional data file.S3 TableEach band was quantified and normalizes by dividing the value from the 0 time and then multiplying the value by 100. (PDF)(PDF)Click here for additional data file.


There is a need to replace petroleum-derived with sustainable feedstocks for chemical production. Certain biomass feedstocks can meet this need as abundant, diverse, and renewable resources. Specific ionic liquids (ILs) can play a role in this process as promising candidates for chemical pretreatment and deconstruction of plant-based biomass feedstocks as they efficiently release carbohydrates which can be fermented. However, the most efficient pretreatment ILs are highly toxic to biological systems, such as microbial fermentations, and hinder subsequent bioprocessing of fermentative sugars obtained from IL-treated biomass.Escherichia coli strains, DH1 and K-12 MG1655, in the presence of two different ionic liquids, 1-ethyl-3-methylimidazolium acetate ([C2C1Im][OAc]) and 1-butyl-3-methylimidazolium chloride ([C4C1Im]Cl). For multiple parallel replicate populations of E. coli, cells were repeatedly passed to select for improved fitness over the course of approximately 40 days. Clonal isolates were screened and the best performing isolates were subjected to whole genome sequencing.To generate strains capable of tolerating residual ILs present in treated feedstocks, a tolerance adaptive laboratory evolution (TALE) approach was developed and utilized to improve growth of two different mdtJI, a multidrug efflux pump, and yhdP, an uncharacterized transporter. Additional mutations were enriched in processes such as transcriptional regulation and nucleotide biosynthesis. Finally, the best-performing strains were compared to previously characterized tolerant strains and showed superior performance in tolerance of different IL and media combinations  with robust growth at 8.5% (w/v) and detectable growth up to 11.9% (w/v) [C2C1Im][OAc].The most prevalent mutations in tolerant clones occurred in transport processes related to the functions of The generated strains thus represent the best performing platform strains available for bioproduction utilizing IL-treated renewable substrates, and the TALE method was highly successful in overcoming the general issue of substrate toxicity and has great promise for use in tolerance engineering.The online version of this article (10.1186/s12934-017-0819-1) contains supplementary material, which is available to authorized users. There is a need to replace chemical and fuel production from fossil feedstocks with carbon neutral sources to retain the natural cycle of carbon emission and assimilation. Certain biomass feedstocks can play a major part in this need as they are abundant, diverse, and renewable. These biomass feedstocks include general plant-based materials like energy crops, crop residues, wood, wood residues, and grasses. Most of these materials have intrinsic value alongside with the added possibility of use as biomaterials . BiomassLignocellulosic biomass can serve as a carbon neutral and abundant feedstock for bioprocesses . In ordeDespite its efficacy, IL pretreatment has some limitations, as some amounts of ILs remain from the pretreatment and these are often highly toxic to microbes used in downstream fermentation processes. Typically, a deconstruction hydrolysate has around 0.2–5% (w/v) of ionic liquid after the pretreatment . One praEscherichia coli (E. coli) strains that were highly tolerant to the presence of ILs. The TALE method differs from previous efforts in that dynamic control is used to increase the amount of stress applied to cells to keep a strong selection pressure without crashing cultures due to overstressed growth conditions. The TALE approach for IL tolerance employed in the present study has two major advantages compared to manual ALE work , to generate k . First,E. coli (K-12 MG1655 and DH1) were exposed to increasing concentrations of two ILs; 1-butyl-3-methylimidazolium chloride and 1-ethyl-3-methylimidazolium acetate. Both of these targeted ILs are promising solvents for biomass pretreatment and were considered as a good candidates for IL-pretreated biomass Cl) was purchased from Sigma-Aldrich , and 1-ethyl-3-methylimidazolium acetate, ([C2C1Im][OAc]), was purchased from IOLITEC ionic Liquids Technologies GmbH . Chemicals and components of the medium used for selecting the best performing strains were purchased from Sigma-Aldrich  or VWR  unless otherwise noted.Two 4, 100 µM CaCl2 and 1× trace elements and Wolfe’s vitamin solution. Composition of 10× M9 salts solution consisted of 68 g/L Na2HPO4 anhydrous, 30 g/L KH2PO4, 5 g/L NaCl, and 10 g/L NH4Cl dissolved in Milli-Q filtered water and autoclaved. M9 trace elements was a 2000× solution containing of 3.0 g/L FeSO4·7H2O, 4.5 g/L ZnSO4·7H2O, 0.3 g/L CoCl2·6H2O, 0.4 g/L Na2MoO4·2H2O, 4.5 g/L CaCl2·H2O, 0.2 g/L CuSO4·2H2O, 1.0 g/L H3BO3, 15 g/L disodium ethylene-diamine-tetra-acetate, 0.1 g/L KI, 0.7 g/L MnCl2·4H2O and concentrated HCl dissolved in Milli-Q filtered water and sterile filtered. The final concentrations of the vitamin mix and trace elements in the M9 medium were 1×.M9 glucose medium contained 2 g/L glucose, 1× M9 salts, 2 mM MgSOE. coli strains, DH1 and K-12 MG1655, were initially screened for their tolerance towards different concentrations of each IL in order to choose the starting concentration where the growth rate and final optical density were higher. A description of tolerance screening and tolerance phenotype in wild type strains  measurements on a sunrise plate reader .The two 600 was measured at a time determined algorithmically and once OD600 reached approximately OD600 0.3, 150 µL was passed into a new tube with a fresh media containing ILs and a total working volume of 15 mL . The commonly experienced exponential growth phase was from time of inoculation to approximately OD600 0.3 and the maximum final OD600 was approximately 0.4, thus the cells were passed during the exponential phase. The OD600 was measured by a Sunrise Plate Reader  and the common ratio between the plate reader OD600 and a benchtop spectrophotometer with a 1 cm path length is 4.2. Growth rates were determined by calculating the slope of the semi-log plot of log OD versus time using linear regression with the Polyfit function in MATLAB . When increased growth rate was achieved after a defined period of time at a particular concentration, the ionic liquid concentration was increased. This process was repeated until a significant increase in tolerance was achieved. Periodically, samples were frozen in a 25% v/v glycerol solution and stored at − 80 °C for further use.The bacterial cells were adaptively evolved under batch fermentation in M9 glucose supplemented with the initial ionic liquid concentration listed in Table 600) on selected concentrations . Populations from endpoint evolutions were plated on LB agar plates and ten individual colonies derived from each population were screened at the maximum concentration for which robust growth rates were achieved during the evolution. Colonies from wild type starting strains, E. coli K-12 MG1655 and DH1, were used as a control for the primary screening. Selected isolates were inoculated into 500 µL M9 glucose medium in deep well plates and incubated in a plate shaker at 37 °C and 300 rpm shaking. Later, cells were diluted 10× in M9 glucose medium, from which 30 µL was transferred to clear-bottom 96 half-deepwell plates  containing M9 glucose medium with either of the two ionic liquids, such that the final concentration was equal to the concentrations listed in Table 600 values using a calibration between OD600 (1 cm path-length) and G values. The resulting growth curves for each isolate, Additional file Evolved isolates were screened for growth properties  in M9 glucose medium with the specified IL concentration, and growth was monitored until stationary phase was reached. Growth rates were calculated as described previously, and the average values of the three cultures were determined.Three individual isolates chosen from primary screening from each population underwent secondary screening, where biological replicates were analyzed. The IL concentration was lowered to the average concentration used in the primary screen for all clones. These adjusted IL concentrations for both ® Genomic DNA Kits . The quality of extracted DNA was assessed with UV absorbance ratios using a nano drop. Concentration of DNA was quantified using Qubit ds-DNA high sensitivity assay. Paired-end resequencing libraries were generated using a 300 cycle (150 bp × 2) kit from Illumina  with loading concentration on Nextseq 1.2 pico-Molar with 1% PhiX spike  of input DNA total. Re-sequencing data were analyzed using a customized script based on the Breseq version 0.30.1 [OAc]  was added to the medium as indicated. Seed cultures in LB medium were grown overnight and diluted 1:50 into M9 glucose medium  containing varying concentrations of [C2C1Im][OAc] or [C2C1Im]Cl . Cultures were grown in 96 well plates  on a Tecan F-200 Pro microtiter plate reader  or in 250 mL flat-bottom flasks at 200 rpm and 37 °C. The absorbance of samples from 250 mL flat-bottom flasks was measured at 600 nm using a Spectramax M2 spectrophotometer . Initially, selected clones were screened in [C2C1Im][OAc] to determine levels of tolerance to a IL not used for the ALE process , then best-performing clones were used for the comparison in [C2C1Im][OAc] and [C2C1Im]Cl in either M9 or LB medium to understand performance in both a minimal defined and rich undefined media. The parent strain E. coli K-12 MG1655 was used as a control. The IL tolerant strains JBEI-10101 Cl) and 1-ethyl-3-methylimidazolium acetate ([C2C1Im][OAc]). The E. coli strains were chosen as they are often used in bioprocessing applications Cl and [C2C1Im][OAc], respectively  and 6.1 ± 0.3% (w/v) for MG1655 and 5.0 ± 0.6% (w/v) and 4.6 ± 0.3% (w/v) for DH1, with [C4C1Im]Cl did not grow; therefore, this population was dropped from the analysis (ALE #8). A total of 45 isolates were whole genome resequenced.Isolates from evolved populations were screened for improved tolerance to ILs and to help identify causal mutations through genotype–phenotype relationships following resequencing. A primary screen was performed to establish whether selected isolates from each population (10 isolates from each of 16 populations) could grow reproducibly in the average final IL concentration achieved during TALE  identified. The MG1655 isolate had 267 mutations, while the DH1 isolates had 39 ± 4 mutations, as compared to the average mutations 5 ± 4 for the non-hypermutating clones from both strains with standard deviation shown between replicates. The hyper-mutator clone from MG1655 had two mutations in two different SOS genes, uvrA and uvrC, which are involved in DNA repair processes under stressed conditions Cl, DH1/[C4C1Im]Cl, MG1655/[C2C1Im][OAc], and DH1/[C2C1Im][OAc] conditions, respectively. Most of the clones showed reproducible performance when culturing. Results for both E. coli K-12 MG1655 and DH1 are summarized in Additional file A secondary screen was performed to generate quantitative data on the resequenced isolates after their genetic bases had been determined see “”. Resequ MG1655/CC1Im[OAc] MG1655/CC1Im[OAc]−1) between isolates that were genetically-identical or genetically-similar were 21 and 11%, respectively. Some of the resequenced isolates exhibited no growth  was detected for all five clones at 8.5% (w/v) IL and detectable growth was observed for the MG clones, MG 4.7, MG 3.10, and MG 4.5, in concentrations up to approximately 11.9% (w/v) (700 mM) [C2C1Im][OAc]  250 mM) [C50 mM [C2rcdA with tolerance up to 3% (w/v) [C2C1Im][OAc] achieved in LB medium. Additionally, thermophilic communities have been isolated by enriching them for tolerance to [C2C1Im][OAc], which has resulted in the identification of a mixed population tolerant to 6% w/v [C2C1Im][OAc] [4C1Im]Cl of approximately 7% (w/v) in rich media (LB) [The final concentration of ILs tolerated by evolved isolates using TALE were compared to previously reported strains generated for IL tolerance Table . The robIm][OAc] . Finallydia (LB) . A direcEnterobacter lignolyticus and its response regulator (eilAR), and the JBEI-13314 [OAc] [5.1% (w/v)] or [C2C1Im][Cl] [4.4% (w/v)]. [C2C1Im][Cl] which contains a chloride anion, was found to be effective towards dissolving cellulose in comparison to larger anions, [C2C1Im][OAc] [2C1Im][OAc]) have lower viscosities and this is beneficial as it facilitates the dissolution process [OAc] . Further2C1Im][OAc] or [C2C1Im]Cl, the performance of JBEI-13314 and JBEI-10101 was improved over the background control of a wild-type MG1655. In the same conditions, the TALE-derived strains, MG 4.7 and MG 3.10, grew at a significantly faster rate and to a higher final density than the rationally engineered strains. Specifically, the TALE derived strain, MG 4.7, grew to a final OD600 of approximately 3.0 and 1.5 in LB containing [C2C1Im][OAc] and [C2C1Im]Cl, respectively, versus final ODs of approximately 1.0 on both ILs for the best performing rationally engineered JBEI-10101 strain. Similarly, the growth rates were higher for the TALE-derived strains  insights into both strain-specific and global mechanisms of IL tolerance through examining key mutations found in multiple parallel evolved isolates, and (3) establishing a viable method using a multiple population TALE approach with next-generation sequencing towards generating tolerant strains. This method was benchmarked via comparison to rational engineering approaches.The economical and efficient break down of lignocellulosic material into carbon feedstocks is an essential step in renewable bioprocessing. Ionic liquid (IL) solubilization is a promising method for breakdown of lignocellulosic material, however these compounds are toxic to most bioproduction chassis strains. Thus, the scope of this study was to generate IL tolerant strains utilizing an adaptive laboratory evolution process. Accordingly, the main contributions from this work are: (1) effective generation of IL tolerant strains (including cross-tolerance) for two common production chassis, 2C1Im][OAc]; detectable growth at 11.9% (w/v) for TALE-derived E. coli K-12 MG1655 clones and robust growth at 8.5% (w/v) for the same MG1655 isolates plus the E. coli DH1 best-performing clones. Thus, the TALE-derived strains show promise as platform strains for utilization of biomass hydrolysates generated using IL treatment.TALE was successful in generating strains that were tolerant to the targeted ILs. After approximately 40 days of continuous exposure to ILs during growth , populations of cells were able to grow at approximately threefold or greater of the initial concentration of each IL compared to the wild type Table . ToleranmdtJ/tqsA intergenic region, as well as in the tqsA gene, and in the yhdP gene ]. The approach used to identify and interpret the key causal mutations using whole genome sequencing complemented with analyzing isolates from multiple independent populations, and multiple isolates from each population, was successful in revealing the key mutations involved in IL tolerance phenotypes. The most striking identified key mutations appeared to involve modulation of transport mechanisms, possibly the direct transport of ILs into and out of the cell. The results of this study and the approach used to generate tolerant strains can be expanded to other conditions, strains, and selection criteria, which would help in fast-tracking the utilization of alternative renewable feedstocks, as well as to better understand tolerance mechanisms for inhibitory compounds.Additional file 1. All of the whole genome sequencing results.Additional file 2. Additional text, tables and figures.