Micro-computed tomography (micro-CT) was applied to elucidate the relationship between the three-dimensional (3D) root surface area (RSA) and two-dimensional (2D) crown-to-root ratio (CRR) of extracted teeth to classify the periodontitis and assign a periodontal/prosthetic prognosis. A total of 31 maxillary and 35 mandibular single-rooted human premolars were examined. The amount of periodontal support on the basis of 3D RSA and 2D root length (RL) at CRRs of 1:1, 5:4, 3:2, and 2:1 were analyzed. Both maxillary and mandibular premolars demonstrated a nonsignificant RSA percentage at the evaluated CRRs. The coronal 21%–22% 2D RL and the 26%–28% 3D RSA bone loss apical to the cemento-enamel junction corresponded to a CRR of 1:1, relating to mild-moderate periodontitis. The coronal 30%–31% 2D RL and the 41%–42% 3D RSA bone loss corresponded to a CRR of 5:4, correlating to severe periodontitis. More severe clinical attachment loss (CAL) was observed in the 3D RSA measurement than in the 2D RL measurement at the evaluated CRRs. The amount of CAL at the CRR of 1:1 was inadequate to assess the severity of periodontitis on the basis of the 2D RL and 3D RSA measurements. Clinicians employ two-dimensional (2D) linear CRR extensively as an objective index to examine whether the candidate’s abutment teeth are indicated for tooth-borne fixed and removable prostheses5. In the field of prosthetics, the CRR is defined as “the physical relationship between the portion of the tooth within the alveolar bone compared with the portion not within the alveolar bone, as determined radiographically”6. An abutment tooth with a higher CRR always involves a lower amount of supporting bone; therefore, it receives a higher loading force from occlusion and an unfavorable lateral force spread from removable partial dentures (RPDs)7. A consensus on the prognostic value of CRR in assigning a prosthetic prognosis is yet to be established9. Dykema10 proposed that a CRR of 1:2 was ideal and that a CRR of 2:3 was acceptable and desirable for fixed partial dentures (FPDs) on abutment teeth. In comparison, CRRs of 2:3 and 1:1 have been reported as the optimal and minimum ratios, respectively, for the abutment teeth on FPDs when the periodontium is healthy and occlusion is controlled12. Moreover, the quantitative evidence to support these values is limited.Dentists evaluate many clinical parameters, such as the amount of bone loss, probing depth, furcation involvement, tooth mobility, root form, pulpal involvement, therapist skill and knowledge, caries, tooth position and occlusal relationship, strategic valve, and crown-to-root ratio (CRR), to assign a prognosis to an individual tooth13. Relying on the CRR value of 1:1, periodontists assign a favorable, cautious, or unfavorable prognosis to a tooth with periodontitis. However, the associations between the optimum CRR and the amount of radiographic bone loss have not been well documented. Furthermore, differentiating satisfactory from unsatisfactory CRR based on the 1:1 ratio to evaluate the diagnosis of mild, moderate, and severe periodontitis is discordant14.The American Academy of Periodontology (AAP) guidelines define mild, moderate, and severe periodontitis as characterized by 15, 16–30%, and >30% radiographic bone loss19. After tooth scanning and software analysis, reconstructed three-dimensional (3D) files can be translated into 2D data for root surface area (RSA) analysis, and information on the RSA of the scanned tooth at the evaluated CRRs can be calculated. We hypothesized that the corresponding amounts of periodontal bone support at the evaluated CRRs based on the 3D RSA measurement are different from those based on the 2D root length (RL) measurement. The purpose of this study was to apply a micro-CT to scan extracted human single-rooted premolars and measure the 3D RSAs at the evaluated linear CRRs levels and to decide if the amount and ratio of the measured 3D RSAs were comparable to those assessed on the basis of 2D RL at same CRRs. The amount of clinical attachment loss (CAL), corresponding to 2D RL measurements, has been applied to diagnose the severity of periodontitis13. The 3D RSA and 2D RL ratios of CALs representing mild, moderate, and severe periodontitis were also analyzed. Finally, the prognostic values of CRR in periodontal and prosthetic appraisals in accordance with the 3D RSA and 2D RL ratios were surveyed and discussed.Micro-computed tomography  has been used to support the clinical findings of dental investigationsA total of 31 maxillary and 35 mandibular premolars were collected from patients undergoing orthodontic or periodontal treatment at the Dental Department of CGMH. The teeth were cleaned and scanned. The attained raw data of tooth structures were obtained as 2D images, which were assembled and converted into 3D reconstructions , total RL was 12.63 ± 0.19 mm (ranging from 10.8 mm to 14.4 mm), and 2D linear anatomical CRR was 1:1.83 ± 0.08 (ranging from 1:1.38 to 1:3.57). For the mandibular premolars, average tooth length was 20.53 ± 0.28 mm (ranging from 17.3 mm to 24.2 mm), 100% RSA was 202.43 ± 4.37 mm2 (ranging from 161.2 mm2 to 277.1 mm2), total RL was 13.28 ± 0.27 mm (ranging from 10.3 mm to 17.0 mm), and anatomical CRR was 1:1.88 ± 0.07 (ranging from 1:1.28 to 1:3.11). A wide distribution pattern was observed for these four examined factors for both premolars; however, significant variation between maxillary and mandibular premolars was observed only for 100% 3D RSA (Table p = 0.002).The results revealed that for the maxillary premolars, average 2D linear tooth length was 19.81 ± 0.31 mm (ranging from 16.4 mm to 23.0 mm), 100% RSA was 226.16 ± 5.99 mm2) reductions of 25.62% ± 1.22%, 40.88% ± 1.65%, 48.67% ± 1.34%, and 60.07% ± 1.06%, respectively, for maxillary premolars and coronal bone support (in mm2) reductions of 28.35% ± 1.23%, 42.35% ± 1.53%, 49.67% ± 1.32%, and 61.15% ± 1.31%, respectively, for mandibular premolars . Maxillary premolars and mandibular premolars demonstrated non-significant differences at the examined levels for both 3D RSAs and 2D CRRs (p ≥ 0.05). The variation between the measured and proposed amounts of bone support also revealed non-significant differences at the examined 2D CRR levels  reductions of 21.41% ± 0.97%, 30.15% ± 0.86%, 37.13% ± 0.77%, and 47.61% ± 0.64%, respectively, for maxillary premolars and coronal root length (in mm) reductions of 22.22% ± 1.0%, 30.86% ± 0.89%, 37.77% ± 0.80%, and 48.15% ± 0.67%, respectively, for mandibular premolars. The reduced percentages of 2D CRRs and 3D RSAs showed significant differences at all evaluated 2D CRR levels (Table p < 0.05). Maxillary and mandibular premolars demonstrated a non-significant difference at the studied levels for both 3D RSA and 2D RL comparisons (p ≥ 0.05). The difference between the measured and proposed amounts of 2D CRRs also revealed a non-significant difference at the studied 2D RL and 3D RSA levels  was considered21, teeth with 2.72–3.02 mm of bone loss at CRR = 1:1 could be diagnosed as having mild periodontitis. Similarly, premolars with coronal 2D RL bone losses of 30.15%–30.86% at a CRR of 5:4 corresponded to the AAP disease classification of severe periodontitis (>30% or >5 mm radiographic bone loss or >5 mm CTA); the diagnosis became moderate periodontitis when 1.0 mm CTA was included in the periodontal support. Measured ratios of 37.13%–37.77% at a CRR of 3:2 demonstrated a nonsignificant variation when compared to the proposed 40% 2D ratio  at CRR = 1:1, the amount of bone loss was associated with the disease classification of moderate periodontitisp < 0.001, Table All 3D RSA and 2D RL percentages significantly differed at the evaluated CRRs  for both types of premolar , a case of mild periodontitis according to the AAP classification should correspond to 15% 2D RL bone loss at the proposed CRR level of 4:5. Inconsistently, 15% coronal 3D RSA bone loss occurred at the proposed CRR level of 3:4 . The risks associated with high radiation doses also restrict the clinical application of micro-CT in routine human physical examinations; conventional 2D radiographs will be continuously indicated for basic dental examination and diagnostics – including periodontal diseases. Other types of teeth, such as incisors, canines, and molars, may demonstrate various CRR characteristics and thus warrant additional studies.Based on the classification of periodontal prognosis provided by McGuireBased on 2D RL and AAP aspects, depending on whether radiographic bone loss is analyzed as a percentage or in terms of millimeters and on whether 1.0 mm CTA is included, a CRR of 1:1 corresponds to mild to moderate periodontitis, a CRR of 5:4 corresponds to moderate periodontitis, and CRRs of ≥3:2 correspond to severe periodontitis. However, when only the amount of radiographic bone loss is considered, CRRs of ≥5:4 are associated with severe periodontitis from a 3D perspective. In addition, a fair prognosis was found for premolars with 2D RL at a CRR of 4:3 and with 3D RSA at a CRR of 1:1 having 25% periodontal attachment and 35% bone support losses. Similarly, CRRs of 3:1 and 2:1 were associated with teeth with questionable prognoses with 50 and 60% periodontal attachments and bone support losses according to the 2D and 3D data, respectively. Taken together, these findings indicate that the systems that are currently applied to determine periodontitis severity and/or to assign periodontal-prosthetic prognoses for dentistry are asymmetrical according to the 2D and 3D viewpoints. Despite the limitations of the present study, we conclude that the 3D RSA and 2D RL measurements of single-rooted human premolars present inconsistent results in terms of the amount of bone support at the evaluated CRRs, regardless of whether CTA is included.Thirty-six extracted and intact human maxillary premolars and 35 mandibular single-rooted premolars were collected from patients with trauma or periodontitis or from patients who underwent orthodontic treatment at the Dental Department of Chang Gung Memorial Hospital (CGMH). This clinical study was conducted in conformity with the Declaration of Helsinki and was approved by the Medical Ethics Committee of Chang Gung Memorial Hospital. All methods were performed in accordance with the Taiwan Dental Association guidelines and regulations. All patients provided written informed consent.Tooth samples were examined from the cuspid to the root apex using micro-CT  based on a cone beam method with the following settings: tube voltage, 100 kV; tube current, 100 µA; pixel matrix, 2,000 × 2,000; slice thickness, 18 μm. The apex of each premolar was stabilized vertically on a fixture that was arranged parallel to the long axis of the tooth. Generally, 20 minutes were required to scan each premolar. Subsequently, files were exported from the micro-CT instrument in digital imaging and communications in medicine and tagged image file data formats, in which a 5-GB file stored 1,300–1,500 slice images obtained for each tooth. These exported data were subsequently analyzed and edited using DataViewer, CTVol, and CTVox software packages . 3D reconstructions were built from 2D images using the software. 3D geometry files were generated for each mask and were saved as stereolithography (STL) files. An STL format model including approximately 1,500,000–2,000,000 fine triangle surfaces describing a 3D premolar structure was developed. The RSAs of premolars were calculated as the sum of specific fine triangle areas using Pro/Engineer software .The 2D RL of a scanned premolar was determined by measuring the distance from the apex to an average CEJ point  were analyzed. In addition, CRRs with 15%, 30%, and 50% coronal 2D RL bone loss and coronal 3D periodontal RSA bone loss were measured and evaluated. The collected teeth were assigned to maxillary and mandibular groups.Both maxillary and mandibular premolars were analyzed after calculating the ratio of the RSA loss at planned levels to the total RSA loss amounts for individual teeth before performing intra- or inter-group analyses to avoid tooth size and root morphology bias.p < 0.05).Independent sample t-tests were conducted to evaluate the significance of differences in general anatomical measurement between the maxillary and mandibular premolars. Independent samples t-tests were also performed to study differences in the 2D RL and 3D RSA bone loss measurements between the maxillary and mandibular premolars at the corresponding CCRs (p < 0.05).Paired t-tests were used to explore the significance of differences between the 2D RL and 3D RSA losses at the evaluated CRRs and 2D RLs, corono-apically | ≤2%). First H1: the values were >2%.Second H0: The variation between the measured and proposed ratios of bone support at linear 2D RLs or 3D RSAs of 15%, 25%, 30%, 35%, 40%, 50%, and 60% were ≤2% | ≤2%). Second H1: the values were >2%.


We developed easy to synthesize and efficient strigolactone analogs with great application potential. Biological activities and receptor binding assays demonstrate the effect of structural modification on the efficacy and specificity of strigolactones. Striga spp., and as branching factors for symbiotic arbuscular mycorrhizal fungi. Sources for natural SLs are very limited. Hence, efficient and simple SL analogs are needed for elucidating SL-related biological processes as well as for agricultural applications. Based on the structure of the non-canonical SL methyl carlactonoate, we developed a new, easy to synthesize series of analogs, termed methyl phenlactonoates (MPs), evaluated their efficacy in exerting different SL functions, and determined their affinity for SL receptors from rice and Striga hermonthica. Most of the MPs showed considerable activity in regulating plant architecture, triggering leaf senescence, and inducing parasitic seed germination. Moreover, some MPs outperformed GR24, a widely used SL analog with a complex structure, in exerting particular SL functions, such as modulating Arabidopsis roots architecture and inhibiting rice tillering. Thus, MPs will help in elucidating the functions of SLs and are promising candidates for agricultural applications. Moreover, MPs demonstrate that slight structural modifications clearly impact the efficiency in exerting particular SL functions, indicating that structural diversity of natural SLs may mirror a functional specificity.Strigolactones (SLs) are a new class of phytohormones that also act as germination stimulants for root parasitic plants, such as  Strigolactones (SLs) are secondary metabolites originally identified as root-derived chemical signals inducing seed germination in root parasitic plants of the family Orobanchaceae . In addiR-configuration) to a structurally variable second moiety  that is connected by an enol ether bridge , the precursor of canonical, orobanchol-like SLs. Orobanchol itself is produced by another rice MAX1 homolog, the orobanchol synthase  . For the synthesis of (E)-methyl 3--2-phenylacrylate (MP3), we used a previously described protocol (H)-furanone  in THF (5 ml) was gradually added. The mixture was stirred at room temperature for 2 h. The reaction mixture was poured into ethyl acetate (70 ml), and organic layer was washed successively with water (80 ml) and saturated sodium chloride solution (80 ml), dried with sodium sulfate, and concentrated in vacuo. Oily residue was purified with silica gel (Wakosil® C-300HG) column with hexane and ethyl acetate as eluent to give title compound MP3 -Methyl 3--2-(4-nitrophenyl) acrylate (MP1) was prepared by nitration of MP3 . These nitrated compounds, 2-nitro (MP2) and 4-nitro (MP1), were separated by a reverse-phase column (ODS) with 1:1 MeOH:water. Physico-chemical properties of MP1 are shown in (n of MP3 . In a 10E)-3--2-(4-nitrophenyl) acrylaldehyde (MP12) was synthesized as follows. To a stirred solution of 4-nitrophenyl acetic acid (1.8 g) in dimethylformamide (5 ml), POCl3  was added slowly (over 15 min) so that the reaction temperature was kept below 70 °C. Then the reaction mixture was stirred at 70 °C for another 12 h. The mixture was poured into 10 g of ice and neutralized by K2CO3 solution (200 ml). The resultant solid was isolated by filtration and dried to give (Z)-3-(dimethylamino)-2-(4-nitrophenyl)acrylaldehyde, which was used in the next reaction without further purification. (Z)-3-(Dimethylamino)-2-(4-nitrophenyl)acrylaldehyde  and 7.7 M NaOH aq 97 μl  was heated at 70 °C until the reaction mixture became homogeneous. Then the reaction mixture was dried by evaporation under reduced pressure. To the solution of resultant residue in DMSO (1.5 ml), 5-bromo-3-methyl-2(5H)-furanone  was added slowly and stirred overnight. The reaction mixture was diluted with diethylether and the organic layer was washed successively with water and saturated sodium chloride solution, dried (Na2SO4) and evaporated  were incubated at 21 °C in amber HPLC vials (adapted from −1) was diluted to the final concentration with methanol (425 μl), water (500 μl) and 25 μl of 1-indanol (1.0 mg ml−1 solution in acetone) as internal standard. The time course of degradation was monitored by UHPLC analysis on a Dionex Ultimate 3000, using a Zorbax Eclipse Plus C18 column . The column was developed at a flow rate of 0.6 ml min–1 at 35 °C with a linear gradient from 5% to 95% acetonitrile in water within 15 min, maintaining the final conditions for another 4 min. The column was operated at 35 °C with a flow rate of 0.25 ml min−1. Compounds eluted from the column were detected with a diode array detector. The relative quantity of remaining (non-degraded) product was determined by comparison with the internal standard. Stability was monitored at 24 h intervals up to 3 weeks.Aqueous solutions of MPs (50 μg mlS. hermonthica and P. ramosa) germination activity was studied as described previously (–5–10–12 M) was applied on each of the six discs, for each concentration. GR24 solutions with equal concentrations and sterile MilliQ water were included as a positive and negative control, respectively. After application, seeds of S. hermonthica and P. ramosa were incubated in dark at 30 °C for 2 d and at 25 °C for 4 d, respectively. Germination (seeds with radicle emerging through the seed coat) was scored under a binocular microscope, and germination rate (%) was calculated.Parasitic weed seeds’  were surface-sterilized by washing with 70% ethanol for 1 min and then with 2.5% sodium hypochlorite for 15 min. Seeds were then rinsed thoroughly with sterile MilliQ water and incubated in water for 2 d at 30 °C in the dark. Pre-germinated seeds were transferred to filter papers containing half-strength MS medium in 90 mm Petri dishes and incubated at 30 °C under fluorescent white light (130–180 μmol m−2 s−1) for 1 week. Seven-day-old seedlings were transferred to 50 ml falcon tubes (one seedling per tube) containing modified half-strength Hoagland nutrient solution and grown in greenhouse. Plants were treated with MPs at 2.5 μM to 2.5 × 10–7 μM, using GR24 as positive control. The compounds were applied six times, twice a week. Number of tillers per plant, plant height and fresh biomass were measured at final harvest.Rice seeds  seedlings were established as mentioned above. Uniform seedlings were transferred to 50 ml tubes containing half-strength Hoagland nutrient solution for 7 d and grown in an incubator under white fluorescent light (130–180 μmol m−2 s−1) with 16 h/8 h (L/D) at 28 °C for 1 week. Leaf segments of 2 cm were cut from middle part of third leaves of rice plants. Each segment was put in a well (in 24-well plates) containing 2 ml of 2.5 mM MES buffer with 0.05% Tween-20, and incubated at 30 °C in the dark for 7 d. After application of MPs, color change, chlorophyll content, ion leakage, and gene expression were monitored daily for 7 d.One-week-old rice  plates supplemented with MPs or GR24 (at 1.0 μM). Plates were stored at 4 °C in darkness for 3 d. To initiate germination, plates were exposed to continuous white light for 24 h, then transferred to continuous monochromatic red light  conditions for another 4 d. For hypocotyl length measurement, at least 30 seedlings were measured using the freely available ImageJ software (http://rsbweb.nih.gov/ij/) after taking digital photographs. A monochromatic red light source was applied as described previously  plates supplemented with GR24 or MPs (at 1.0 μM). Plates were stored at 4 °C in darkness for 3 d and then vertically grown at 22 °C in a Percival incubator under long day condition  for 8 d. For determining the effect on lateral root density and primary root length, at least 30 seedlings were measured using ImageJ software after scanning roots.Sterilized Arabidopsis seeds were sown on half-strength MS  cDNA, GenBank accession AY097402, was synthesized and cloned into pUC57 (GenScript). ShHTL7 and OsD14 cDNAs were amplified by RT-PCR using the primers shown in BamHI and XhoI, and ligated into BamHI/XhoI-digested pGEX-6P-1 expression vector . Integrity of plasmids was confirmed by sequencing (KAUST Bioscience Core Lab). The plasmids were then transformed into E. coli BL21 (DE3) cells. The cells were grown in LB broth containing ampicillin (100 mg ml−1), incubated at 37 °C until OD600 of 0.6 and induced with 0.1 mM isopropyl-β-D-thiogalactopyranoside at 16 °C for 16 h. Harvested cells were resuspended in lysis buffer: 50 mM Tris–HCl (pH 8.0), 200 mM NaCl, 0.5% Tween-20 and 2 mM dithiothreitol (DTT). After sonication on ice for 10 min, the lysate was centrifuged at 75 000 g for 30 min at 4 °C. Supernatant was allowed to bind to glutathione–sepharose beads  for 2 h at 4 °C, washed 3 times with buffer (50 mM Tris-HCl (pH 8.0), 200 mM NaCl, 2 mM DTT), and eluted by cleaving the GST moiety with PreScission™ Protease  at 4 °C overnight. Eluted protein was further purified by gel filtration using a HiLoad 16/60 Superdex200 prep grade column  on an AKTA prime system  in gel filtration buffer . Protein eluted as a sharp single peak and purity was judged by 4–15% SDS-PAGE (Bio-Rad). Purified protein was concentrated using an Amicon 10K filter unit (Merck Millipore) and stored at −80 °C until use. A similar expression and purification protocol was followed for OsD14 protein, except that OsD14 was eluted from glutathione resins using 0.2 mM reduced glutathione (Sigma-Aldrich) without cutting the GST tag.Kd) values were calculated by fitting to a binding saturation single-site model with Prism 6 (GraphPad).ShHTL7/D14 tryptophans were excited at 280 nm and emission intensity was measured at 333 nm. ShHTL7/D14 (10 μM) was incubated at various dilutions  of GR24 and MPs for 30 min at room temperature before measurement. Each concentration point was measured in triplicate. Emitted fluorescence was monitored using a spectraMaxi3 plate reader (Molecular Devices) in 96-well black plates. Changes in tryptophan fluorescence intensity occur due to conformational changes in protein when it is bound to ligand; differences in fluorescence intensity were recorded and analysed. Data were normalized and dissociation coefficient (−1 and incubated for the indicated time at 37 °C. After adding 1-indanol (10 µl of a 2.5 mg ml−1 solution in methanol), as internal standard, solutions were filtered and transferred to HPLC vials. Hydrolysis of substrates was monitored by HPLC analysis using a Zorbax Eclipse Plus C18 column , eluted by a gradient from 10% to 90% acetonitrile in water within 15 min, keeping the final condition for 4 min. The column was operated at 30 °C with a flow rate of 0.2 ml min−1. Eluted compounds were detected using a diode array detector. Amounts of remaining substrates were determined by calculating corresponding peaks, in comparison with an internal standard.The hydrolysis of GR24 and MPs by ShHTL7/OsD14 was performed in a total volume of 0.5 ml of phosphate-buffered saline containing 10 µM of substrate. Purified ShHTL7/OsD14 was added at a concentration of 50 µg ml50) were calculated to determine optimum amount of MPs. The synthetic strigolactone analog GR24 was used as reference to estimate efficacy of selected compounds. EC50 and estimated response were calculated using the drc package (https://cran.r-project.org/web/packages/drc/) with a four parameter logistic curve (dose–response curve) . Dose–response curves and half-maximum effective concentrations (ECe curve) .We synthesized 14 methyl phenlactonoates (MPs) as described above in ‘Material and methods’  for S. hermonthica seed germination, using concentrations ranging from 10–5 to 10–12 M. Among MPs, we observed the highest activity with MP1, which exhibited an EC50 value of 1.5 × 10–9 M. Other MPs were at least ~5-fold weaker, showing EC50 values ranging from 3.2 × 10–8 (MP2) to 1.7 × 10–6 M (MP11). The EC50 of the lead compound, MP3, was 8.7 × 10–8 M. GR24 was the most active analog, exhibiting an EC50 value of 9.1 × 10–11 M  plants; this was reduced to one tiller per plant in all MP- and GR24-treated samples  in treated and untreated samples , followed by MP7 (2.54 × 10–8 M), GR24 (4.83 × 10–2 M) and MP1 (2.42 × 10–1 M)  . This re 10–1 M) . Besides 10–1 M) . We assuOsl20 (coding for branched chain α-keto dehydrogenase) and Osl295 (coding for aspartic protease)  , E.We also tested the effect of MPs on hypocotyl elongation in Arabidopsis seedlings. Apart from MP12 and MP15, all MPs showed significant inhibition of hypocotyl growth, particularly MP7, which displayed a stronger inhibitory effect than GR24 that was applied at the same concentration 1.0 μM; . We alsoS. hermonthica seeds, we determined the affinity of ShHTL7 for MP1, MP3, and MP7 by measuring its intrinsic tryptophan fluorescence upon binding to different concentrations of these compounds and in comparison with GR24. ShHTL7 exhibited the highest affinity towards GR24 with a Kd value of 0.44 ± 0.21 µM  among the MPs tested here. This activity was around 17 times weaker than that of GR24, pointing to MP1 as a suitable candidate for the suicidal germination approach. Very recently, we have evaluated the activity of MP1 in a trial conducted in Burkina Faso, in which we used 1 × 1 m2 wooden boxes filled with artificially infested soil and pearl millet as a host. In this experiment MP1 showed significantly higher germination activity than GR24 (to be published). The activity of MP1 in inducing seed germination in P. ramosa was quite similar to that of GR24 and only slightly weaker than that of MP2, which carries the nitro group at the ortho position, and of the chlorophenyl compound MP8. Apart from these three compounds, we observed lower germination activity with P. ramosa seeds than with those of S. hermonthica.Previously, we designed a carlactone-based analog, nitro-phenlactone, that showed very weak activity in stimulating d10/CCD8 mutant, we used a hydroponic culture system  and MP7 (EC50=2.54 × 10–8 M) are more efficient than GR24 (EC50=4.83 × 10–2 M) in restoring wild type tillering to the d10 mutant. MP1, which showed the highest activity in the S. hermonthica seed germination test, was weaker than GR24. We also observed that the treatment with several MPs (at 2.5 μM), particularly MP3, leads to growth retardation and senescence. Therefore, we tested the activity of MP3 in accelerating dark-induced leaf senescence, as reported for SLs before  followed by MP9 (ortho position) and MP8 (meta position).Comparison of the activities of different MPs unraveled the impact of substitutions in the phenyl ring on the efficiency of these compounds in exerting specific SL functions. The lead compound, MP3, was less active than MP1 in inducing S. hermonthica SL receptor ShHTL7 for these compounds. The results obtained were consistent with the S. hermonthica germination assay, with GR24 being the most active ligand followed by slightly weaker MP1, MP7, and finally MP3. However, we observed higher hydrolysis activities with MP3 and MP7 than with GR24. We also compared these compounds regarding their affinity and hydrolysis by the rice SL receptor D14. MP1 exhibited the highest hydrolysis rate and the lowest Kd, followed by GR24, MP3, and finally MP7. It may be speculated that the high conversion of MP1 and MP7 is due to the presence of electron withdrawing groups NO2 (MP1) and Cl (MP7), which alleviate the hydrolysis by the two receptors. In the case of ShHTL7 assays, hydrolysis and affinity results are consistent with the determined biological activity. In contrast, MP3 was the less preferred substrate in the incubations with OsD14, although it showed the highest activity in the tillering assay. This difference indicates that other factors, such as uptake and transport, are also decisive for the tillering inhibitory and growth retarding activity. However, it is also possible that hydrolysis rates determined here do not accurately reflect the situation in planta where D14 is part of a protein complex that may impact or modulate hydrolysis activity of this receptor.To shed light on molecular events underlying the differences in activity between MP1, MP3, and MP7, we determined the affinity of Our findings showed that MPs are highly efficient SL analogs that can be used to investigate the biological functions of SLs and employed to combat root parasitic weeds or to modulate plant architecture. Moreover, our study demonstrated that type and position of substitutions in the phenol ring, which corresponds to the A-ring in SLs, determine and modulate the efficiency of MPs in exerting specific SL functions.JXB online.Supplementary data are available at Fig. S1. Stability analysis of MPs in comparison with GR24.Fig. S2. Parasitic seed germination in response to MP treatment.Fig. S3. Effect of MPs on rice fresh biomass. −7 μM, on tillering and growth of d10 seedlings.Fig. S4. Images showing the effect of selected MPs  and GR24, applied at concentration ranging from 2.5 μM to 2.5 × 10Fig. S5. Effect of MPs on Arabidopsis hypocotyl length. Table S1. Methyl phenlactonoates: physico-chemical properties.Table S2. List of primer sequences used in the study.Table S3. Yield and quantity of synthesized MPs.erx438_suppl_supplementary_figures_s1_s5_tables_s1_s3Click here for additional data file.The supplementary tables and figures are also available at the Dryad Data Repository: htpp://dx.doi.org/10.5061/dryad.hq7b8Striga and tillering bioassays, studied leaf senescence, and wrote the manuscript. BAK helped in parasitic seed and tillering bioasays, data compiling and analysis. IH, STA, and USH conducted ShHTL7/OsD14 binding, hydrolysis studies. XG did stability analysis. VON conducted leaf senescence and SAG gene expression studies. KJ studied Arabidopsis hypocotyl length and lateral root inhibition. SA studied Arabidopsis primary root length. YL, JK, KH, and MT prepared and purified protein and carried out the hydrolysis. TA synthesized MPs and SAB designed and proposed the MP study.MJ designed experiments, performed


Aims. In this study a streptozotocin induced type 1 diabetes mouse model was used to assess the effectiveness of salsalate, menhaden oil, the combination of salsalate and menhaden oil, or resolvin D1 on neuropathic endpoints. Materials and Methods. Changes in body weight, blood glucose, serum markers for triglycerides, free fatty acids, cholesterol, and resolvin D1, motor and sensory nerve conduction velocities and thermal sensitivity were assessed, as well as performing in vivo confocal microscopy of subepithelial corneal nerves and immunohistochemistry of nerves in the cornea and foot pad. Results. Diabetic animals failed to gain weight and had elevated blood glucose levels. Diabetic mice had slowed nerve conduction velocity, reduced innervation of the foot pad and cornea subepithelial and epithelial layers, and reduced thermal sensitivity. Monotherapy treatment with salsalate, menhaden oil, and resolvin D1 reduced the pathological signs of diabetic neuropathy. The combination of salsalate and menhaden oil also reduced signs of pathology and generated elevated plasma levels of resolvin D1 compared to other groups. Conclusions. Additional studies are needed to determine whether the combination of salsalate and menhaden oil may be more efficacious than monotherapy alone for the treatment of diabetic peripheral neuropathy. Diabetic peripheral neuropathy occurs in over 50 percent of diabetic patients. It is associated with foot ulceration and is the leading cause of nontrauma foot amputations . ApproveConsumption of fish oil was shown to benefit those with cardiovascular diseases like congestive heart failure and has shown promise in type 2 diabetes, inflammatory diseases, stroke, depression, and anxiety disorders –8. The iSalsalate is a prodrug of salicylate and its primary mechanism of action is as a nonsteroidal anti-inflammatory that functions by inhibiting the synthesis of prostaglandins by way of inactivation of cyclooxygenase enzymes . ExperimIn this study we have used a streptozotocin induced type 1 diabetes mouse model, which we previously characterized , to asseChemicals used in this study came from Sigma-Aldrich chemical company , unless otherwise stated.C57Bl/6J mice were purchased from Jackson Laboratories and housed in a certified animal care facility with food and water available ad libitum. Measures were taken to minimize the number of animals and their pain or discomfort. Experiments were conducted in accordance with international standards on animal welfare complying with institutional and National Institutes of Health animal use guidelines (ACURF protocol 1212258).Twelve-week-old C57Bl/6J mice were divided into six groups, one vehicle treated control group and five groups treated with streptozotocin as previously described , 23. MicThermal nociceptive response was assessed in mice one week prior to euthanasia using the Hargreaves method with an IITC Life Science Inc. device  as previously described , 24. FivMice were anesthetized with Nembutal  and motor and sensory nerve conduction velocities were assessed as in previous experiments , 23, 24.2.The Rostock cornea module for the Heidelberg Retina Tomograph  was used for in vivo assessment of subepithelial nerves in the mouse cornea as described previously , 23. Aneβ-III tubulin mouse monoclonal antibody at 1 : 1000 in 4°C . Following three 15-minute washes with PBS containing 0.2% Triton X-100, goat anti-mouse IgG2a Alexa 488 secondary antibody was applied for 2 hours at room temperature . Three more 15-minute washes in PBS containing 0.2% Triton X-100 preceded the mounting of the cornea (epithelium facing up) to a microscope slide in ProLong Gold . A cover slip was placed on the cornea and sealed with clear fingernail polish.During dissection the animal's cornea was isolated for immunofluorescent analysis as previously described , 23. Theμm × 3400 μm × 30 μm; 4096 pixels × 4096 pixels × 30 pixels) images of the entire mouse cornea that were further processed to make a maximum projection intensity image for analysis. These images were analyzed for the amount of surface area covered by nerves using Imaris version 7.6.4 X64 software . The surface option was used  to determine the total surface area covered by nerves and is represented as a percentage of the total cornea surface area determined by manually tracing the cornea with a closed poly-line, as in previous experiments [μm × 405 μm × 30.11 μm; 1536 pixels × 1536 pixels × 78 pixels) was taken with optimum axial resolution to image the epithelial nerves of the cornea. These images were cropped to include only the epithelial nerves and subjected to volume analysis using Imaris version 7.6.4 X64 software ; the nerve volume is represented as a percentage of total volume as used for previous experiments [Using Zeiss LSM710 Confocal Microscope running ZEN software  multiple images were collected to assess different nerve parameters in an attempt to more fully characterize the diseased cornea. Firstly, a 20x objective (Plan-Apochromat 20x/0.8) was used to make 8 × 8 tile scan z-stack . One-way analysis of variance (ANOVA) and Sidak's multiple comparisons test were performed to determine group differences . F = 14.54, p < 0.0001). Throughout the experiment, diabetic animals had elevated blood glucose levels that were greater than in control mice and were not significantly impacted by treatment  = 60.49, p < 0.0001 and F = 12.14, p < 0.0001, resp.).Animals were diabetic for a total of 20 weeks and for the final 12 weeks selected animals received treatment via dietary supplementation of menhaden oil, salsalate, or menhaden oil plus salsalate or daily injection of 1 ng/g resolvin D1. As seen in Serum triglycerides and free fatty acids were not significantly changed in any of the groups.F = 3.53, p < 0.01; diabetic group  26.8 ± 1.4, diabetic plus salsalate group 28.6 ± 4.0, and resolvin D1 group 27.3 ± 1.1 mg/dL). For mice receiving menhaden oil and menhaden oil plus salsalate serum cholesterol was increased over the control group .Serum cholesterol levels were 20.0 ± 1.2 mg/dL in control mice but trended towards an increase in all diabetic groups  = 8.13, p < 0.0001; menhaden oil 862 ± 130 pg/mL, menhaden oil plus salsalate 1151 ± 117 pg/mL, and control 680 ± 52 pg/mL) while the untreated diabetic group (diabetic group 613 ± 47 pg/mL) remained unchanged from control, salsalate, and resolvin D1 groups .As seen in F = 16, p < 0.0001). Following treatment with menhaden oil, menhaden oil plus salsalate, and resolvin D1 subepithelial corneal nerve occupancy appears similar to the control mice  whereas salsalate treatment alone had marginal but significant improvement over diabetic animals (1.8 ± 0.6 and 1.0 ± 0.4 mm/mm2). Using an antibody to β-III tubulin we further examined the density of corneal nerves in the epithelial and subepithelial layers by immunohistochemistry. F = 4.4, p = 0.0015; 49 ± 2.7 and 61 ± 2.2% area, resp.) and treatment with salsalate, menhaden oil, and menhaden oil plus salsalate provides a trend towards improvement while resolvin D1 treatment significantly increases nerve surface area over diabetic animals . As observed in the representative images untreated diabetic mice show reduced subepithelial nerve bundle length and compared to control mice a greater area of the cornea is devoid of β-III tubulin staining.The in vivo analysis of corneal nerves with corneal confocal microscopy is presented in F = 14, p < 0.0001). Treatment with menhaden oil, menhaden oil plus salsalate, or resolvin D1 provided significant benefits compared to untreated diabetic mice  and salsalate alone showed a modest nonsignificant increase over untreated diabetic mice (1.1 ± 0.1% volume).F = 12.47, p < 0.0001, and control 41.1 ± 1.5 versus diabetic group 27.7 ± 1.0 m/sec; SNCV F = 21.49, p < 0.0001, and control 29.9 ± 0.7 versus diabetic group 22.5 ± 0.6) and treatment with menhaden oil, menhaden oil plus salsalate, and resolvin D1 produced significantly faster conduction velocities compared to untreated diabetic mice   . TreatmeF = 44.20, p < 0.0001; 14.6 ± 0.4, 24.7 ± 0.7, 18.4 ± 0.4, 21.3 ± 0.5, 21.0 ± 0.6, and 20.4 ± 0.3 profiles/mm, resp.). All diabetic mice showed a reduced thermal sensitivity compared to control animals, except those treated with resolvin D1  = 32.0, p < 0.0001; control 5.7 ± 0.2 versus diabetic group 8.6 ± 0.2 sec). Treatment with salsalate, menhaden oil, menhaden oil plus salsalate, and resolvin D1 significantly improved thermal sensitivity over untreated diabetic mice .Untreated diabetic mice show a reduction in intraepidermal nerve profiles compared to control mice and treatment with salsalate, menhaden oil, menhaden oil plus salsalate, and resolvin D1 showed significantly more nerve profiles , the subsequent reduction in NF-κB related genes, and increased nitric oxide (NO) preventing ischemic nerve damage [In humans, salsalate has been used to treat the pain and inflammation associated with rheumatoid arthritis, osteoarthritis, and other rheumatic disorders , 31. Theycerides , 19, 32.ycerides  with impycerides , 20. Somycerides  and redue damage , 36. ThiFish oils, like menhaden oil, contain a high concentration of EPA and DHA and their consumption elevates the plasma concentration of proresolving inflammatory lipid mediators like resolvin D1 . This stThere are at least two ways to produce resolvin D1 from DHA. DHA may be acted upon by 15-lipoxygenase, peroxidases, 5-lipoxygenase, and hydrolases before the derivate resolvin D1 is generated. Additionally, aspirin-triggered resolvin D1 can be generated by acetylated cyclooxygenase followed by activity of a peroxidase and hydrolase . We prevIn this study, when animals received the combined treatment of salsalate and menhaden oil we did not observe an improvement in diabetic neuropathy outcome measures beyond monotherapy with menhaden oil. This was not surprising since the study was not designed to examine the potential for elevating lipid mediator levels following low dose fish oil combined with salsalate treatment. In this study we used the maximum concentration of menhaden oil possible for treatment of type 1 diabetic rodents . And whiThe principle finding of our study is that anti-inflammatory compounds such as salsalate and dietary supplements such as fish oil can provide benefit to patients suffering from diabetic peripheral neuropathy. Whether the combination of salsalate and lower doses of fish oil may provide a similar benefit for treatment of diabetic neuropathy as the higher doses of fish oil will require further investigation.


CH2Cl2 is not a common dopant due to its high ionization energy (11.33 eV). The low-pressure photoionization source was built using a krypton VUV lamp that emits photons with energies of 10.0 and 10.6 eV and was operated at ~500–1000 Pa. Protonation of water, methanol, ethanol, and acetaldehyde was respectively enhanced by 481.7 ± 122.4, 197.8 ± 18.8, 87.3 ± 7.8, and 93.5 ± 35.5 times after doping 291 ppmv CH2Cl2, meanwhile CH2Cl2 almost does not generate noticeable ions itself. This phenomenon has not been documented in the literature. A new protonation process involving in ion-pair and H-bond formations was proposed to expound the phenomenon. The observed phenomenon opens a new prospect for the improvement of the detection efficiency of VUV photoionization.Doping has been used to enhance the ionization efficiency of analytes in atmospheric pressure photoionization, which is based on charge exchange. Compounds with excellent ionization efficiencies are usually chosen as dopants. In this paper, we report a new phenomenon observed in low-pressure photoionization: Protonation enhancement by dichloromethane (CH Photoionization (PI), a widely used soft ionization technique, is usually coupled to various mass spectrometers for analyzing the chemical composition of samples12346Low-pressure photoionization (LPPI), defined as photoionization running under hundreds to thousands of Pa, has not been used as widely as APPI and conventional vacuum PI. LPPI has characteristics of both vacuum PI and APPIwhere A represents the analyte molecules and R is the reagent which offers a proton or hydrogen atom. The reagent could be the analyte or solvent molecules.The use of dopants has been found to be very effective for enhancing the ionization efficiency of analytes679103OH, IE = 10.84 eV), ethanol , and acetaldehyde  due to their higher IEs. Dichloromethane has been chosen as a dopant for characterizing the molecular structures of analytes via secondary ion–molecule reactions, rather than for enhancing ionization efficiencywhere D and A represent dopant and analyte molecules, respectively. Benzene (IE = 9.24 eV)131015166710111218193OH, C2H5OH, and C2H4O is very low due to their low ionization efficiencies. In this paper, we report a new phenomenon: The detection efficiencies of the three small volatile organic compounds (VOCs) can be remarkably enhanced via CH2Cl2 doping. The results and experimental method are described in the following sections.Our previous studies revealed that LPPI with a specially designed photoionizer was super sensitive (~1000 counts/ppbv) to many organic compounds24252O) is an important protonation agent for PTR mass spectrometry. The IE of water is 12.62 eV, which indicates that it cannot be photoionized directly by the photons emitted from the krypton lamp. However, H3O+ , (H2O)2H+ , and (H2O)3H+  were observed in the LPPI mass spectrum of N2, as shown in 2 gas. Protonation of acetonitrile (IE = 12.20 eV) was observed in APPI with a krypton lamp as the VUV light source by Marotta et al. The authors speculate that photon irradiation leads first to the isomerization of acetonitrile molecules, affording species that exhibit IEs <10 eV and that consequently are able to generate photoionization products2+  observed in 2Cl2 into the chamber. Surprisingly, the signal intensities of H3O+, (H2O)2H+, and (H2O)3H+ increased to 2.92 × 104, 1.24 × 105 and 2.29 × 104 counts, respectively. The signal intensity of protonated water was averagely enhanced by 481.7 ± 122.4 times, measured from three independent measurements. This phenomenon has never been reported.Water  is the simplest alcohol. Its IE is 10.84 eV, higher than the energy of the photons emitted from the krypton lamp. A weak signal of protonated methanol was observed when 4.6 ppmv methanol was sampled. 3O+, (H2O)2H+, and (H2O)3 H+, respectively. The mass peaks at m/z 33, 51 and 65 are assigned to (CH3OH)H+, (CH3OH·H2O)H+ and (CH3OH)2H+, respectively. The moderate mass peak at m/z 47 is assigned to ethanol, the impurity in the methanol reagent. The peak intensities of (CH3OH)H+ and (CH3OH)2H+ are 559 and 171 counts, respectively. It is reported in the literature that dimers of methanol (CH3OH)2 with IE equal to 9.72 eV coexist with methanol monomers under ambient conditions and that protonated methanol is generated from the dissociation of (CH3OH)2+282Cl2. The signal intensities of the mass peaks at m/z 33 and 65 reach 1.48 × 105 and 6.06 × 104 counts, respectively. The signal intensity of protonated methanol was averagely enhanced by 197.8 ± 18.8 times, measured from three independent measurements.Methanol (CH2H5OH) is 10.48 eV, meaning it can be photoionized by the photons emitted from the krypton lamp . 2H5O+ (551 counts), (C2H5OH)H+ (1923 counts), and (C2H5OH)2H+ (222 counts). The mass peak of protonated ethanol was the strongest peak. After doping with 291 ppmv CH2Cl2, the intensities of the mass peaks at m/z 47 and 93 shown in 5 and 2.21 × 104 counts, respectively. The signal intensity of protonated ethanol was averagely enhanced by 87.3 ± 7.8 times, measured from three independent measurements. The mass peak at m/z 45 slightly increased to 2765 counts, while the mass peaks at m/z 29 (1.54 × 104 counts) and 65 (1.80 × 104 counts) assigned to C2H5+ and (C2H5OH·H2O)H+ appeared.The IE of ethanol (C2H4O) is one of the most important aldehydes; it occurs widely in nature and is produced industrially on a large scale. The IE of acetaldehyde is 10.23 eV. 2H4O)H+ (1290 counts), and (C2H3O·H2O)+ (1307 counts), respectively. The molecular ion of acetaldehyde was not observed. Protonated acetaldehyde was the dominant ion. 2Cl2. The signal intensity of protonated acetaldehyde (m/z 45) increased to 7.04 × 104 counts, while the signal at m/z 61 slightly increased to 2107 counts. The signal intensity of protonated acetaldehyde was averagely enhanced by 93.5 ± 35.5 times, measured from three independent measurements. Additionally, a mass peak at m/z 63 assigned to (C2H4O·H2O)H+ (1.71 × 104 counts) appeared.Acetaldehyde (C6H6) is an important chemical and atmospheric pollutant. Its IE is 9.24 eV, lower than the energy of VUV photons emitted from the krypton lamp. Benzene and its derivatives have excellent photoionization efficiencies under illumination of a krypton VUV lamp. 12C6H6+ (6.42 × 104). 2Cl2. The intensities of the mass peak at m/z 78 decreased by ~14% to 5.54 × 104 counts. The fluctuation of the signal intensities at m/z 78 was observed in separate experiments. No signal enhancement at m/z 79 (protonated benzene) was observed in all experiments.Benzene  and are excited to Rydberg states302 does not have absorptionet al. reported that ion-pair states were observed in halogenated methanes excited by VUV light and ion pair states even existed below ionization potentials3 and CH2Cl2, excited by the krypton lamp, may form the ion-pair states of [H+−CCl3−] and [H+−CHCl2−], which facilitate protonation. Other dopants including H2, CH4, and CCl4 do not meet the combined conditions of formation of ion-pair states and existence of H atoms.Pure CH2Cl2 doping:+−CHCl2−] represents an ion-pair state, [A−H+−CHCl2−] sketches a complex formed via a H bond, and A denotes analyte molecules, i.e. molecules of water, methanol, ethanol, and acetaldehyde. The proposed scenario of protonation enhancement is as follows: 1. CH2Cl2 excited by VUV light transforms into an ion-pair state ([H+−CHCl2−]); 2. The analyte molecule collides with [H+−CHCl2−] and forms a complex [A−H+−CHCl2−] via a H bond; 3. The detachment of the proton from CH2Cl2 leads to the formation of a protonated analyte molecule (AH+) and CHCl2−. This hypothesis rationalizes all the experimental observations. To the best of our knowledge, protonation via collision with excited-state molecules has not yet been documented. The heat of reaction (ΔrH°) of deprotonation of CH2Cl2 (CH2Cl2 = CHCl2− + H+) is ~16.3 eVwhere [HThe experimental setup has been described in detail elsewhereThe 120 L test chamber was mainly built with an open-head stainless steel drum and covered with a thin Tedlar bag to ensure one atmospheric pressure during sampling. A stainless steel fan driven by a magnetic field was placed at the bottom of the test chamber to ensure quick mixing. Nitrogen was used as the buffer gas. An oil-free pump was used as the drain pump. Two mass flow controllers were used for gas samples. All experiments were performed under ambient atmospheric pressure and room temperature.2 window. The optical baffle was placed at the exit of the photoionization source to prevent the VUV light entering the mass spectrometer. The LPPI source was passivated with ~600 ppm CH2Br2 under illumination of VUV light for ~8 hours to suppress photoelectron formation in the experiment. The krypton lamp was laboratory-assembled and emitted VUV photons with energies of 10.0 eV (~80%) and 10.6 eV (~20%). The sample gas was introduced into the photoionization source and controlled by a needle valve. The sample flow was maintained at ~1 cm3 s−1. The pressure in the photoionization source was 500–1000 Pa. The mass spectrometer was a simple V-shaped time-of-flight mass spectrometer with a free-field flight distance of 460 mm. The cycle time of detection was 10 s.The LPPI mass spectrometer was recently developed in our laboratory. It characterizes with a LPPI source with an optical baffle and a short reflectron time-of-flight mass spectrometer. The body of the LPPI source was a cylindrical stainless steel cavity 6 mm in diameter and 35 mm in length. A radio frequency-driven krypton VUV lamp was used as the VUV light source and coupled to the cylindrical stainless steel cavity with an MgF2Cl2 was injected into the test chamber and the mass spectra were subsequently acquired after each injection. The amount of methanol, ethanol, acetaldehyde, and benzene injected into the nitrogen-filled test chamber was 1.0, 0.5, 0.5, and 0.2 μL, respectively. The resulting mixing ratios for the prepared gases were 4.6, 1.6, 0.66, and 0.42 ppmv, respectively. The injection of 100 μL CH2Cl2 resulted in 291 ppmv in the mixing ratio.In the experiments, a small amount of bottle-contained chemical was first injected into the test chamber. Then, 100 μL CH2Cl2 , CHCl3 , and CCl4  were used. High-purity nitrogen (>99.99%), hydrogen (>99.999%), and methane (>99.9%) were purchased from Beijing Huayuan Gas Co., Ltd.In this study, methanol , ethanol , acetaldehyde , benzene , CHHow to cite this article: Shu, J. et al. Protonation enhancement by dichloromethane doping in low-pressure photoionization. Sci. Rep.6, 36820; doi: 10.1038/srep36820 (2016).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Myostatin is small glycopeptide that is produced and secreted by skeletal muscle. It is a potent negative regulator of muscle growth that has been associated with conditions of frailty. In C2C12 cells, myostatin limits cell differentiation. Myostatin acts through activin receptor IIB, activin receptor-like kinase (ALK) and Smad transcription factors. microRNAs (miRNA) are short, 22 base pair nucleotides that bind to the 3′ UTR of target mRNA to repress translation or reduce mRNA stability. In the present study, expression in differentiating C2C12 cells of the myomiRs miR-1 and 133a were down-regulated following treatment with 1 µg of recombinant myostatin at 1 d post-induction of differentiation while all myomiRs  were upregulated by SB431542, a potent ALK4/5/7 inhibitor which reduces Smad2 signaling, at 1 d and all, with the exception of miR-206, were upregulated by SB431542 at 3 d. The expression of the muscle-enriched miR-486 was greater following treatment with SB431542 but not altered by myostatin. Other highly expressed miRNAs in skeletal muscle, miR-23a/b and 145, were altered only at 1 d post-induction of differentiation. miR-27b responded differently to treatments at 1 d, where it was upregulated, as compared to 3 d, where it was downregulated. Neither myostatin nor SB431542 altered cell size or cell morphology. The data indicate that myostatin represses myomiR expression in differentiating C2C12 cells and that inhibition of Smad signaling with SB431542 can result in large changes in highly expressed miRNAs in differentiating myoblasts. •Myostatin down-regulates the myomiRs in myoblasts prior to and during myotube formation.•Inhibition of ALK signaling increases expression of highly-expressed miRNA in myoblasts.•Myostatin and ALK inhibition has a greater effect on the miRNA profile at 1 D post-induction of differentiation. Pre-miRNAs are then exported out of the nucleus by exportin-5 and further processed into an active form by Dicer Due to the deleterious effects of myostatin on muscle, it is not surprising that it is associated with conditions of frailty and muscle wasting Knowledge of the role of myostatin in the regulation of miRNAs in skeletal muscle development and homeostasis remains limited. Transcriptome analysis using deep sequencing supports a role for myostatin in regulating miRNA expression. Inhibition of myostatin in a transgenic mouse model overexpressing the myostatin propeptide revealed 57 differently expressed miRNAs as compared to wild-type littermates We have observed that SCI is associated with decreased expression of miR-23a/b, miR-27b, miR-145 and miR-206; a pathways analysis implicated these miRNAs as being known or predicted to repress translation of multiple proteins involved in TGFβ family signaling Thus, there is ample evidence that miRNAs expressed in skeletal muscle regulate the expression of multiple key proteins in myostatin/ACRRIIB/Smad2/3 signaling as well as other signaling pathways that govern muscle protein catabolism and muscle atrophy and hypertrophy programs. The objective of the present study was to determine whether myostatin could regulate the expression of muscle-specific and highly expressed miRNAs that were identified as being differentially regulated in gastrocnemius muscle after SCI 22.1Mouse C2C12 myoblasts were grown to confluency in 6-well plates in proliferating media consisting of Dulbecco’s Modified Eagle’s Medium (DMEM) with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S). At confluency, the media was removed and cells were washed twice in 1x phosphate buffered saline (PBS) and covered with differentiating media consisting of DMEM supplemented with 2% horse serum and 1% P/S. Cells were harvested at 1 day (1 D) and 3 days (3 D) post-induction of differentiation. One day prior to cell harvest, media was supplemented with vehicle (4 mM HCl with 0.1% BSA), recombinant myostatin (R&D Laboratories) at 100 ng/ml and 1 µg/ml without or without co-incubation with 1 µM SB431542  and 1 µM SB431542 alone. SB431542 is a small molecule that selectively inhibits ALK 4, 5 and 7 2.2Morphology of C2C12 cells was determined after seeding cells on 2-well glass culture slides  under the same conditions used above. When the incubation was concluded, the slides were rinsed three times in 1x PBS, fixed for 15 min in 4% PFA and stored covered in 1x PBS at 4 °C until analysis. Cells were imaged using an EVOS FL Auto Cell Imaging System  and analyzed using either the EVOS software or ImageJ . Variables analyzed were nuclear counts normalized to protein content as measured by microBCA obtained for cells used for western immunoblotting, and cell area. Nuclei were visualized by staining with DAPI and imaged by fluorescence microscopy. Nuclear counts were determined by stitching together a 3×3 grid of 10x images and averaging the nuclear count of each stitched image then expressed as nuclei per nanogram protein. Average cell area was calculated by tracing 300 cells per condition (100 per well) using a brightfield image obtained at 20x magnification using the trace tool in ImageJ.2.3g for 15 min at 4 °C. The clear supernatant fraction was collected and RNA, including miRNA, was isolated and collected with a column according to the manufacturer’s instructions . RNA was quantified using a Nanodrop 1000 . All samples had 260/280 ratios over 1.9. 1 µg of RNA was mixed with an RT primers pool (Applied Biosystems) for all of the miRNAs for the current study and reverse transcribed at 16 °C for 30 min, followed by a 30 min step at 42 °C and finished with a 5 min step at 85 °C . qPCR was conducted with gene expression assays for miR-1, miR-23a/b, miR-27b, miR-133a/b, miR-145, miR-206 and miR-486 using specific TaqMan miR assays . The default qPCR protocol was used, specifically, a 10 min holding step at 95 °C, then 40 cycles of a 15 s step at 95 °C followed by a 60 s step at 60 °C. Individual ΔΔCts were calculated by using the equation:Cells were lysed with 700 µL of Qiazol , placed into 1.5 ml microcentrifuge tubes and homogenized by vigorous vortexing. Chloroform (140 µL) was added and the solution was phase-separated by centrifugation at 14,000 – .U6 RNA was used as the housekeeping gene. At the 1 and 3 d timepoints, respectively, the U6 Cts for the experimental groups (mean +/- SD) were as follows: Con=21.87+/- 0.15 and 21.73+/- 0.17; 100 ng/ml myostatin=21.62+/- 0.21 and 21.79+/- 0.12, 100 ng/ml myostatin + SB431542=21.77+/- 0.18 and 21.87+/- 0.13; 1 µg/ml myostatin=21.84+/- 0.19 and 21.66+/- 0.14; 1 µg/ml + SB431542=21.68+/- 0.14 and 21.73+/- 0.13; SB431542=21.84+/- 0.06 and 21.81+/- 0.19. Fold-change for each sample was calculated as 2 raised to the power of the -ΔΔCt. 2.4Cells were lysed with 1x RIPA buffer supplemented with protease and phosphatase inhibitors. The supernatant was collected and analyzed for protein content with a microBCA kit . Lysates (30 µg of protein) were mixed 1:1 in Laemlli sample buffer and boiled for 3 min. Protein was then separated using a 10% polyacrylamide gel and transferred to a PVDF membrane. The membrane was stained with a Ponceau S solution to visualize protein loading and destained with 0.1 M NaOH. Membranes were quickly rinsed in Tris-buffered saline with 0.05% Tween 20 (TBS-T) and placed in a blocking solution of 5% bovine serum albumin (BSA) in TBS-T for an hour and incubated at 4 °C overnight with the primary antibody in 1% BSA/TBS-T solution. An antibody that detected both phosphorylated serine(465/467) Smad2/serine(423/425) Smad3 (#8828) and total GAPDH  were used in a 1:1000 dilution while MyoD  was used at 1:500. After the overnight incubation, membranes were rinsed in TBS-T and placed in a 1:2000 solution of a horseradish peroxidase-conjugated secondary antibody  and 1% milk and TBS-T for an hour at room temperature. Membranes were rinsed in TBS-T and incubated with a horseradish peroxidase chemiluminescent substrate  for 5 min and imaged using a digital imager. The bands were quantitated with ImageQuant software  and normalized to total GAPDH.2.5Due to the limited effects of 100 ng/ml of myostatin on miRNA expression, an additional 3 wells was studied to confirm that this dose was largely ineffective. Thus, n=6 for gene expression studies for the vehicle and 100 ng/ml of myostatin groups and n=3 for all other experiments was used. All statistical analyses were completed using Prism 7.0 statistical software . Two-way ANOVAs (Treatment × Time) were used to detect interaction effects at p<0.05 for gene expression and morphology. Follow-up analyses for simple means differences were tested with one-way ANOVAs and Sidak’s multiple comparisons tests. The effect of myostatin and SB431542 on protein expression, as well as significant main effects, were analyzed with one-way ANOVAs at each time point with Sidak’s multiple comparisons tests when appropriate. Significance was noted when p<0.05.33.1Treatment of cells with myostatin or pharmaceutical inhibitors of Smad activation may alter cell size and morphology as well as myoblast differentiation. This possibility was evaluated by examining cell size, protein content, and morphology. Examination of cells by brightfield microscopy revealed that fusion of myoblasts had not occurred under any of the conditions tested and that morphology was similar for cells treated with vehicle, myostatin or SB431543 C-D. Howe3.2To confirm that myostatin and SB431542 altered Smad activation under the conditions of the experiments, as has been previously reported in the literature,  levels o3.33.3.1In low serum conditions, C2C12 myoblasts exit the cell cycle and begin to differentiate. There were significant treatment × time interaction effects in regards to the expression of the canonical myomiRs as well as the muscle-enriched miR-486 . Overall3.3.2Incubation with 100 ng/ml myostatin did not alter levels of miRs 23a/b, 27b or 145 at 1 or 3 D A-D. At 14The above studies aimed to determine whether myostatin modulated expression of a selected set of miRNAs in differentiating C2C12 myoblasts. The miRNAs included three miRNAs that are downregulated in muscle after SCI and are known to target mRNA transcripts encoding proteins involved in signaling by myostatin and other TGFβ family members  The reduction of pSmad2 after adding SB431542 suggests that there is activation of signaling through Smad2/3 under basal conditions, possibly as a result of myostatin or other TGFβ family members in the horse serum added to the growth media. At one day after inducing differentiation, effects of myostatin on miRNA-expression were dose-related; the (100 ng/ml) dose of myostatin did not alter expression of any of the miRNAs examined whereas the higher dose (1 µg/ml) suppressed miR-1 and miR-133a at 1 but not 3 D, and also reduced miR-23a, miR-23b and miR-145. The repression of miRNA expression by myostatin at 1 D was at least partially blocked by the ALK4/5/7 inhibitor. In addition, at 1 D, the inhibitor virtually eliminated pSmad2 and elevated expression of miRs 1 and 23a/b. The time after initiating differentiation was a determinant of the response to myostatin or the ALK4/5/7 inhibitor suggesting that in some way, the cellular context  is important to myostatin and ALK/4/5/7 signaling and their effects on miRNA expression. Specifically, the pattern of the changes elicited by myostatin or the ALK4/5/7 inhibitor at 3 D were similar to those at 1 D for miR-1 and miR-133a and miR-486, though not identical, while changes in miR-27b expression induced by these treatments were completely different at 1 and 3 D. The above findings support the view that under some conditions at least, myostatin represses expression of at least two miRNAs that target expression of proteins involved in signaling downstream of myostatin: miR-23a and miR-145. One might propose therefore a feed-forward amplification of myostatin signaling via repression of the expression of these miRNAs.There were, however, some discordant findings in our data, such as the finding that both myostatin and the ALK4/5/7 inhibitor elevated miR-486 expression, which cannot be explained by the action of myostatin signaling through ALK4/5 alone, and which presumably reflect the action of other TGFβ family members with distinct actions on miRNA expression that act through parallel pathways involving either ALK4/5 or ALK7.Alterations in miRNA expression induced by myostatin and ALK4/5/7 may have other effects on muscle homeostasis as well. Transcription of miR-1, miR-133a/b and miR-206 generally follows similar patterns during muscle differentiation, in part because genes for these miRNAs are found in two clusters, the miR-1/133a cluster and the miR-206/133b cluster miR-23a/b has been implicated in muscle atrophy because it targets two muscle-restricted E3 ligases, MuRF1 and MAFbx, which are involved in the breakdown of muscle proteins during atrophy induced by diverse conditions including denervation and glucocorticoid administration Ank1 gene, the gene from which miR-486 is derived Our findings regarding the myomiRs are largely in agreement with Rachagani et al. miR-27b can target the 3′-UTR of Pax3, reduce Pax3 levels and drive the differentiation program of muscle cells in vitro and in vivo, as well as miR-23a overexpression in transgenic mice, prevented dexamethasone-induced muscle atrophy in vivo or ex vivo would be anticipated to shed more light on the question of the influence of myostatin on expression of these miRNAs in skeletal muscle.The influence of myostatin and the ALK4/5/7 inhibitor on miRNAs expression has broader implications. miR-23a and miR-23b differ by only a single nucleotide and target two E3 ubiquitin ligases known to participate in skeletal muscle atrophy, MuRF1 and MAFbx


Purpose: Asian populations have around 40–60% frequency of reduced function allele ∗10CYP2D6 compared to 1–2% in Caucasian populations. The wide range of CYP2D6 enzyme activities in subjects with the ∗10CYP2D6 variant is a big concern for clinical practice. The quantitative analysis measuring the impact of CYP2D6 enzyme activity as a result of one ∗10CYP2D6 allele or two ∗10CYP2D6 alleles has not been reported in large Asian populations.Methods: A total of 421 healthy Chinese subjects were genotyped for CYP2D6 by polymerase chain reaction and direct DNA sequencing. A total of 235 subjects with ∗1/∗1CYP2D6 (n = 22), ∗1/∗10CYP2D6 (n = 93), ∗10/∗10CYP2D6 (n = 85), and ∗5/∗10CYP2D6 (n = 35) were phenotyped for CYP2D6 using dextromethorphan as the probe drug. Metabolic ratios (MR) were calculated as the ratio of parent drug to metabolite in 0–3 h urine, 3 h plasma, and 3 h saliva for each sample type.Results: The urinary, plasma, or salivary MRs increased successively in subjects with ∗1/∗1CYP2D6, ∗1/∗10, ∗10/∗10, and ∗5/∗10 . In the normal metabolizer group, homozygous ∗10/∗10CYP2D6 decreased the CYP2D6 enzyme activity further than heterozygous ∗1/∗10CYP2D6. Urinary, plasma, and salivary MRs were highly correlated.Conclusion: The normal metabolizer group calls for a more detailed classification. The activity score system could more accurately predict enzyme activity than by grouping a number of genotypes into a single phenotype group. Single-point plasma samples and saliva samples could be used as alternative phenotyping methods for clinical convenience. CYP2D6 gene encodes the cytochrome P450 2D6 enzyme that is a member of the cytochrome P450 superfamily. It plays an important role in the metabolism of approximately 25% of currently marketed drugs. This enzyme participates in metabolizing a number of substrates in the therapeutic class of clinical drugs including antidepressants, antipsychotics, analgesics and antitussives, beta adrenergic blocking agents, antiarrhythmic, antiemetics, etc.  from urine samples  to its metabolite dextrorphan (DX) is employed to differentiate between NMs and PMs (CYP2D6 phenotyping.Phenotypes are stratified into groups including poor (PM), intermediate (IM), normal (NM), and ultra-rapid (UM) metabolizer phenotypes . The act and PMs . However and PMs . The MR  and PMs ,b. The M and PMs . These r∗10CYP2D6 variant is a reduced function allele that decreases enzyme activity and thus increases the CYP2D6 MR value. However, quantitative analysis on the impact of CYP2D6 enzyme activity as a result of one ∗10CYP2D6 allele or two ∗10CYP2D6 alleles has not been reported in large populations. In the present study, ∗1CYP2D6 was defined as a standard full functional allele and ∗5CYP2D6 was defined as a non-functional allele. Both ∗1CYP2D6 and ∗5CYP2D6 were used as references or control alleles to investigate the impact of the ∗10CYP2D6 allele on the metabolic activity of CYP2D6 in a healthy Chinese population. The CYP2D6 enzyme activities within the population were determined simultaneously by urinary, plasma, and salivary phenotyping methods and the results were compared.It has been shown that the The study was performed in accordance with the Declaration of Helsinki and was approved by the Ethics Committee of Peking Union Medical College Hospital. Written informed consent was obtained from each subject. Four hundred and twenty-one healthy unrelated subjects in Mainland China were enrolled. All enrolled subjects were judged to be healthy based on the results of detailed physical examination, 12-lead electrocardiography, biochemistry, hematology, and routine urinalysis. Subjects were not eligible if they had history or evidence or hepatic, renal, gastrointestinal, or hematologic abnormality; hepatitis B or C, syphilis, or human immunodeficiency virus infection on screening examination; any other acute or chronic disease; or allergic to dextromethorphan. The consumption of alcohol, grapefruit juice, and caffeine-containing drinks was not permitted for 24 h prior to DM administration and until all samples were collected in the study phase. The subjects were instructed to abstain from taking any medication or herbal remedies for at least 1 week and smoking for at least 3 days before the study.∗1, ∗2, ∗3, ∗4, ∗6, ∗7, ∗10, ∗14, ∗18, ∗21, ∗28, ∗33, ∗34, ∗35, ∗36, ∗39, ∗41, ∗43, ∗49, ∗51, ∗52, ∗54, ∗60, ∗63, ∗65, ∗69, ∗71CYP2D6, and ∗75 as previously reported  . The DNA from 2 mL of blood was dissolved in 100 μL of DNA hydration solution and stored at -70°C. All subjects recruited in this study were genotyped by DNA sequencing analysis for reported . Long porocedure . CYP2D6 ∗1/∗1CYP2D6 (n = 22), ∗1/∗10CYP2D6 (n = 93), ∗10/∗10CYP2D6 (n = 85), and ∗5/∗10CYP2D6 (n = 35) were phenotyped for CYP2D6 using DM as the probe drug and DX as the CYP2D6-specific metabolite. Each subject received 15 mg DM  with 300 ml of water. Venous blood samples and saliva samples were collected 3 h post-drug administration. Urine samples were collected at 0–3 h intervals post-drug administration. Concentrations of DM and unconjugated DX in all samples were analyzed using a sensitive and validated high performance liquid chromatography tandem mass spectrometry (HPLC–MS/MS) assay as previously described (DM/DX) was used as a measure of CYP2D6 enzyme activity in the three sample types, respectively.A total of 235 subjects with escribed . The low∗1CYP2D6 was defined as a standard full functional allele and ∗5CYP2D6 was defined as a non-functional allele. Both ∗1CYP2D6 and ∗5CYP2D6 were used as references or control alleles to estimate the impact of the ∗10CYP2D6 allele on CYP2D6 metabolic activity. The AS system was measured by assigning scores of 2, 1.5, 1, and 0.5 to subjects with ∗1/∗1CYP2D6, ∗1/∗10, ∗10/∗10, and ∗5/∗10, respectively. A P value less than 0.05 was considered to be statistically significant. The ANOVA was performed with SPSS .Data were expressed as mean values ± SD. Based on urinary, plasma, and salivary phenotyping methods, the MR values between different genotype groups were analyzed respectively with an analysis of variance (ANOVA) test. Allele Table 1.Of the investigated subjects, 404 subjects were ethnically Han; the remaining 17 subjects included Hui, Manchu, Khalkhas, Korean, Zhuang, Tujia, and Mongol ethnicities. Their ages, weights, body mass index (BMI) and genders can be found in ∗10CYP2D6 was 45.7%. There were 22 subjects with ∗1/∗1CYP2D6, 93 subjects with ∗1/∗10CYP2D6, 85 subjects with ∗10/∗10CYP2D6, and 35 subjects with ∗5/∗10CYP2D6. The ∗10CYP2D6 allele was also heterozygous with other alleles besides ∗1 and ∗5 although those genotypes were not reported in this study. Other CYP2D6 alleles were tested to determine ∗1, ∗5CYP2D6, and ∗10 specifically and remove the confounding factors.Among the 421 subjects, the gene frequency of Based on the MR values from the 0 to 3 h urine samples, the mean MR in 235 subjects was 0.485 ± 2.48, ranging from 0.00758 to 36.7, and displayed greater than 4,800-fold inter-individual range. Based on the MR values from the 3 h plasma samples, the mean MR in 235 subjects was 1.73 ± 6.63, ranging from 0.0528 to 95.6, and displayed greater than 1800-fold inter-individual range. Based on the MR values from the 3 h saliva samples, the mean MR in 235 subjects was 5.24 ± 18.8, ranging from 0.117 to 250, and displayed greater than 2100-fold inter-individual range.∗10CYP2D6 allele had substantial impact on the metabolic activity of CYP2D6 regardless of the urinary, plasma, or salivary phenotyping method used. The mean (± SD) MRs for ∗1/∗1, ∗1/∗10, ∗10/∗10CYP2D6, and ∗5/∗10 groups were presented by the three methods, respectively . The urinary, plasma, or salivary MRs increased successively in subjects with ∗1/∗1, ∗1/∗10, ∗10/∗10CYP, and ∗5/∗10 with statistical significance  (Table 2).The P < 0.001) . A statistically significant correlation was also found between urinary MR and salivary MR and the Spearman’s correlation coefficient was 0.717 (P < 0.001) . Similarly, the correlation between plasma MR and salivary MR was also statistically significant and the Spearman’s correlation coefficient was 0.903 (P < 0.001) .A statistically significant correlation was found between urinary MR and plasma MR. The Spearman’s correlation coefficient measuring the statistical dependence between the ranking of two variables was 0.780  in the Chinese Han population, followed by ∗1CYP2D6 (26.5%)  . It is w∗1/∗1, ∗1/∗10, ∗10/∗10CYP2D6, or ∗5/∗10 . According to the Clinical Pharmacogenetics Implementation Consortium (CPIC) guideline, subjects with ∗1/∗1CYP2D6, ∗1/∗10, and ∗10/∗10 are classified as NMs, and subjects with ∗5/∗10CYP2D6 are classified as IMs (P < 0.001). In the NM group, however, a large diversity in MR values was still observed. Although ∗1/∗10CYP2D6 and ∗10/∗10CYP2D6 were both classified as NMs similar to the designation given to ∗1/∗1CYP2D6, the enzyme activity for subjects carrying ∗10CYP2D6 differed to a great extent from the wild type due to the reduced function allele. Both ∗1/∗10CYP2D6 and ∗10/∗10 had predicted different enzyme activities as compared with ∗1/∗1. Regardless of which phenotyping method was used, the MRs of subjects with ∗10/∗10CYP2D6 were significantly higher than those of subjects with ∗1/∗10CYP2D6 and ∗1/∗1CYP2D6, respectively, and the MRs of subjects with ∗1/∗10CYP2D6 were significantly higher than those of subjects with ∗1/∗1CYP2D6 . Homozygous ∗10/∗10CYP2D6 could decrease the CYP2D6 enzyme activity further than heterozygous ∗1/∗10CYP2D6. The mean MR was about 4.6-fold higher in subjects with ∗10/∗10CYP2D6 than in subjects with ∗1/∗10CYP2D6 based on the urinary phenotyping method, and the corresponding folds were 5.8 for the plasma method and 4.8 for the salivary method. In comparison, the mean MR was about 4.7-, 2.8-, and 3.9-fold higher in subjects with ∗5/∗10CYP2D6 than in subjects with ∗10/∗10CYP2D6 based on urinary, plasma, and salivary phenotyping methods, respectively. The difference between ∗1/∗10CYP2D6 and ∗10/∗10 was comparable or even larger than that between ∗10/∗10CYP2D6 and ∗5/∗10 and can be seen in Figure 1. Large overlaps were observed between ∗10/∗10CYP2D6 and ∗5/∗10 as shown in Figure 2. Therefore, the NM group calls for a more detailed classification as the large diversity in the NM group could result in an inaccurate prediction of CYP2D6 enzyme activity. As discussed previously, the ∗10/∗10CYP2D6 would be more appropriately classified as IM than NM. Our additional analysis showed that two subjects with ∗5/∗5CYP2D6 had a plasma MR ratio > 70 suggesting that the ∗5/∗10 individuals are distinct from PMs.The urinary, plasma, or salivary MR values increased successively in subjects with d as IMs . In the CYP2D6. It has since gained acceptance among the scientific community and has been adopted by CPIC . The AS system could further classify the NM group into AS 2, 1.5, and 1. On the other hand, the diversity of enzyme activity was observed when evaluating the AS system in Caucasian populations. ∗1CYP2D6 at 1.0. The ∗2 and ∗35 alleles were also assessed as NM alleles. The ∗2 allele had an estimated scaled activity of 0.63 while the ∗35 allele had an activity of 1.03 . Urinary MR appeared to have a smaller correlation coefficient than plasma and salivary MRs. This trend was also seen in previous studies  caused the wild type number with two full functional alleles to be relatively small in the present study. The frequency of PM is very low (0–2%) in the Chinese population . In the NM group, homozygous ∗10/∗10CYP2D6 showed a greater decrease in CYP2D6 enzyme activity than the heterozygous ∗1/∗10CYP2D6 genotype. The NM group calls for a more detailed classification due to its broad range of MR values. The AS system has shown to be a more accurate measurement for enzyme activity prediction compared to grouping a number of genotypes into a single phenotype group. Urinary, plasma, and salivary MRs were highly correlated and the latter two could be used as alternative phenotyping methods for clinical convenience.The urinary, plasma, or salivary MR values increased successively in subjects with RC designed and preformed the study. XZ analyzed the samples. PH designed the study.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


FMR1 gene that triggers epigenetic gene silencing. Epigenetic modifying drugs can only transiently and modestly induce FMR1 reactivation in the presence of the elongated CGG repeat. As a proof-of-principle, we excised the expanded CGG-repeat in both somatic cell hybrids containing the human fragile X chromosome and human FXS iPS cells using the CRISPR/Cas9 genome editing. We observed transcriptional reactivation in approximately 67% of the CRISPR cut hybrid colonies and in 20% of isolated human FXS iPSC colonies. The reactivated cells produced FMRP and exhibited a decline in DNA methylation at the FMR1 locus. These data demonstrate the excision of the expanded CGG-repeat from the fragile X chromosome can result in FMR1 reactivation.Fragile X syndrome (FXS) is a common cause of intellectual disability that is most often due to a CGG-repeat expansion mutation in the  FMR1 gene where, in contrast to the common normal repeat length of 30 triplets, FXS alleles expand well beyond 200 triplets [FMR1 alleles with this expanded repeat are referred to as the full mutation. In a response to the expanded repeat, the FMR1 gene undergoes locus-specific hypermethylation and chromatin remodeling that epigenetically silences the gene [FMR1.Fragile X syndrome (FXS) is the most common cause of inherited intellectual disability and is one of the leading monogenic causes of autism . FXS is the gene . Althougthe gene , 7, the the gene , 8–12, othe gene –15. RegaFMR1, strategies to reactivate FMR1 in FXS cells have been attempted. Small molecules that inhibit DNA methyltransferases [FMR1 transcription. However, such reactivation is typically only transient, and re-silencing of FMR1 happens within days. Moreover, long-term use of such inhibitors imposes serious deleterious effects on the cells, which is potentially due to unnecessary expression of other genes caused by these small molecules.Since the CGG-repeat is in the 5’UTR and would not influence the coding region of sferases , 17 or tsferases , 5 have A site specific genomic editing tool, the CRISPR  system, has recently been developed and implemented to target and mutate specific genomic regions–21. The FMR1 allele without CGG-repeats. We hypothesized that excision of the expanded CGG-repeat from the FMR1 full mutation may lead to constant FMR1 reactivation. Initially, to ease cell culture and enhance cell cycling, we utilized a somatic hybrid cell line (Y75) that contains a single human fragile X chromosome in a rodent CHO cell line [FMR1 transcription and translation in about half of the excised colonies. Similarly, we implemented the CRISPR experiments in human induced pluripotent stem (iPS) cells derived from a FXS patient [FMR1 in fragile X syndrome.In this work, we utilized CRISPR genome editing technology to excise the expanded CGG-repeat from the full mutation allele in FXS cells resulting in an ell line . Success patient . After sFMR1 into the backbone vector PX458 containing the GFP and Cas9 cassettes to construct two plasmids, named SW59 and SW60 (We first designed and cloned a pair of single guide RNA (sgRNA) oligos targeting either side of the CGG repeat tract of and SW60 . Cells tand SW60 . To valiand SW60 .FMR1 allele  [FMR1.Not all CRISPR-cut colonies exhibited ne (5mC) . After tFMR1. However, they induced a single double strand break (DSB) near the CGG repeat and relied upon somewhat random nonhomologous end joining to excise the repeat. Thus, the precision of the CRISPR deletion was not as precise as our approach of creating two flanking DSB. Similar to our observations, Park et al. also observed reactivation of FMR1, although differences in experimental details make frequency comparisons between the two studies difficult. However, the results presented above validate that CRISPR deletion of the full mutation CGG repeat on the fragile X chromosome can reactivate the silenced FMR1 gene.While this work was being concluded, Park et al. (Cell Rep Oct 13 2015) reported similar work using CRISPR excision of the full mutation with reactivation of HEK293FT was maintained in DMEM supplemented with 10% FBS and 1% P/S at 37°C with 5% CO2 incubation. Somatic hybrid cell lines Y75 and Y130 were maintained in HAS medium  at 37°C with 5% CO2 incubation. iPSCs were maintained in the feeder-free culture system following the protocol from STEMCELL Technologies. iPSCs were fed daily with mTeSR1  on matrigel  coated plate and were passed every 5–7 days using the enzymatic free method with ReLeSR .6 cells were seeded into one well of a 6-well plate one day prior to transfection. 2 μg CRISPR plasmids and 2 μl P3000 was diluted in 125 μl opti-MEM  and was further mixed with 4 μl lipofectamine 3000 diluted in 125 μl opti-MEM for each well to be transfected. After 5 min incubation at room temperature, the transfection reagent mixture was added evenly into the well. 48–72 h post transfection, cells were ready to be harvested for other downstream experiments.HEK293FT cells were transfected using Lipofectamine 3000 . Specifically, 0.5 x 106 cells were seeded into one well of a 6-well plate one day prior to transfection. 1h prior to transfection, complete HAS medium was switched to antibiotic-free HAS medium (2 ml/well). 2 μg CRISPR plasmids diluted in 250 μl opti-MEM was mixed with 4 μl LTX diluted in 250 μl opti-MEM for each well to be transfected. After 25 min incubation at room temperature, the transfection reagent mixture was added evenly into the well. 24 h post transfection, cells were ready for FACS. 48–72 h post transfection, cells were ready to be harvested for other downstream experiments.Somatic hybrid cell line Y75 was transfected using Lipofectamine LTX . Specifically, 0.2 x 106 iPSCs resuspended in human stem cell nucleofector solution 1 were nucleofected with 6 μg CRISPR plasmids (SW59+SW60) using program B16 in the nucleofector IIb device . After being transferred to a freshly coated matrigel plate, the nucleofected cells were fed with mTeSR1 containing 10 μM Y-27632 for 1 d followed by regular mTeSR1 medium.iPSCs were transfected using nucleofection method. Specifically, 1 d prior to the passage day, iPSCs were treated with 10 μM Y-27632 overnight. On passage day, 4 x 10™ DNA extraction solution  following the manufacturer’s protocol. We use 100 μl or 400 μl of the solution for each well of a 24-well or 6-well plate, respectively. For Southern blot, DNA was extracted using QIAGEN DNeasy Blood & Tissue Kit  following the manufacturer’s protocol.For genotyping, DNA was extracted using Epicentre QuickExtractTaq DNA polymerase . 2 μl DNA template in Epicentre QuickExtract™ DNA extraction solution was mixed with 12.5 μl 2x GC buffer I, 1 μl 10 μM primer CRISPR Del_F (5’-GGAGGGAACAGCTTGATCAC-3’), 1μl 10 μM primer CRISPR Del_R (5’-ACTGGACTTGGGGCCTGTT-3’), 4 μl dNTP mixture, 0.25 μl LA Taq and 4.25 μl nuclease-free water to a final volume of 25 μl, and further subjected to a thermocycler program as follows: 94°C for 5 min,  x 35 cycles, 72°C for 5 min, 4°C hold. PCR products were analyzed on a 1.5% agarose gel.The CRISPR cut allele was amplified using TaKaRa LA FMR1 qPCR. Genotyping positive colonies were further seeded into a 96-well plates at the density of 0.5 cell/well to isolate morphologically single colonies. After confirming purified single colonies by genotyping, colonies were viewed as pure CRISPR cut clonal lines.Transfected Y75 cells sorted on a Becton Dickinson FACS Aria II cell sorter were first seeded into a 24-well plate at the density of 5 cells/well. 7 days after the seeding single or mixed colonies would appear. 14 days after the seeding cells were ready to make replica plates for genotyping and human specific 4 cells/cm2. iPS cell aggregates appear between d7 and d10 post sorting. At this time point, pour a new layer of MEF to coat the plate again to substitute the old MEF layer. Continue feeding the cells until iPSC colonies were big enough for toothpick PCR based CRISPR deletion genotyping. Deletion positive colonies were transferred to the matrigel plate and cultured as pure CRISPR cut clonal lines.Transfected iPS cells sorted on a Becton Dickinson FACS Aria II cell sorter were first seeded onto a MEF layered 24-well plate at the density of 100 cells/well. The MEF coating density was 4 x 10A total of 8 μg genomic DNA was fast digested with 5 μl EcoRI, 5 μl PteI, 5 μl 10x buffer and nuclease free water in a 50 μl reaction system at 37°C for 2 h. The digested DNA fragments were separated on a 0.8% agarose gel and transferred to nylon membrane. Prehybridize the membrane with 600 μl salmon sperm and 14 ml hybridization buffer  at 65°C for 3–4 h. During prehybridization, prepare p32 labeled probe (primers for amplifying probes are listed in 5’-AGAGGACAAGGAGGAAGAGGACGT-3’), 0.6 μl 10 μM primer hs_FMR1 qPCR R (5’-CTTTACCCGTGCGCAGCCGAC-3’), 0.25 μl iScript reverse transcriptase, 4.55 μl nuclease-free water to a final volume of 20 μl and further subjected to a thermocycler program in Bio-Rad CFX96 as follows: 50°C for 10 min, 95°C for 1 min,  x 40 cycles, 95°C for 1 min, 55°C for 1 min, melt-curve analysis from 55°C to 95°C with 0.5°C increment/10 s. Control primers are listed in RNA was extracted using Trizol LS  following manufacturer’s protocol. iTaq universal SYBR Green one-step kit  was used for human specific qPCR assay. 4 μl RNA template (50 ng/μl) was mixed with 10 μl 2x iTaq universal SYBR Green reaction mix, 0.6 μl 10 μM primer hs_FMR1 qPCR F  at 200 V for 30 min, and transferred to PVDF membrane using Trans-blot Turbo Transfer System  at 1.3 A, 25 V for 10 min. Membrane was blocked with StartingBlock T20 (PBS) blocking buffer  at room temperature for 15 min followed by incubating with human specific anti-FMRP primary antibody (6B8)  or anti-eIF4E primary antibody  at 4°C overnight. The next day, the membrane was washed with blotto buffer  for 5 min at room temperature followed by incubation with anti-mouse secondary antibody for 1 h at room temperature. After 3 times of 10 min washes with blotto buffer at room temperature, the membrane was incubated with the clarity western ECL substrate solution  for 5 min and exposed with the ChemiDoc Touch imaging system . Image analysis was performed using Image Lab™ Software .Cells were lysed in 50–100 μl lysis buffer per well of a 6-well plate for 30 min on ice. Lysate was clarified by centrifugation at 14000 rpm for 10 min. A total of 30–40 μg protein for each sample was separated on 4–15% Mini-PROTEAN TGX Stain-FreeFMR1 sequence used for experimental design and alignment was from Human Assembly Feb. 2009 (GRCh37/hg19) at position: chrX:146992469–147032647.Sequencing alignment was done using CLC sequence viewer 7.0.2. Human S1 Fig(PDF)Click here for additional data file.S2 Fig(PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.


The solar cell performance was optimized by changing the N doping contents. The power conversion efficiency of solar cells based on N-TiO2 with the N doping content of 1%  has been achieved 11.1%, which was 14.7% higher than that of solar cells based on un-doped TiO2. To get an insight into the improvement, some investigations were performed. The structure was examined with X-ray powder diffraction (XRD), and morphology was examined by scanning electron microscopy (SEM). Energy dispersive spectrometer (EDS) and Tauc plot spectra indicated the incorporation of N in TiO2 nanorods. Absorption spectra showed higher absorption of visible light for N-TiO2 than un-doped TiO2. The N doping reduced the energy band gap from 3.03 to 2.74 eV. The photoluminescence (PL) and time-resolved photoluminescence (TRPL) spectra displayed the faster electron transfer from perovskite layer to N-TiO2 than to un-doped TiO2. Electrochemical impedance spectroscopy (EIS) showed the smaller resistance of device based on N-TiO2 than that on un-doped TiO2.In this paper, N-doped TiOThe online version of this article (doi:10.1186/s11671-016-1811-0) contains supplementary material, which is available to authorized users. The par cells , 8. Compar cells , 10. Thear cells , 12. Howlar cell .2 as a photoanode of dye-sensitized solar cells (DSSCs) can improve the energy conversion efficiency due to the change of properties of TiO2, such as electron lifetime prolongation, charge transfer resistance reduction, and visible light absorption extension [In order to solve the issues, some methods have been adopted, such as metal doping , 15 and xtension , 18.2 on the performance of perovskite solar cells. Hence, in the present study, we synthesized N-doped TiO2 (N-TiO2) nanorod arrays with hydrothermal method and fabricated perovskite solar cells using them as electron transfer layer. The solar cell performance was optimized by changing the N doping contents. The PCE of solar cells based on N-TiO2 with the N doping content of 1%  has been achieved 11.1%, which was 14.7% higher than that of solar cells based on un-doped TiO2. The possible mechanisms of enhancement were discussed based on some investigations.We wondered about the effect of N-doped TiO2 compact layer was deposited by dipping the substrate in a 0.2 M TiCl4 aqueous solution at 70 °C for 30 min. TiO2 NRs were grown on the treated FTO substrate by a hydrothermal method in our previous report [2)2 was added to the solution  and stirred until it was completely dissolved. The FTO substrate was put into the solution and sealed in an autoclave. The autoclave was heated to 170 °C for several hours. The obtained TiO2 nanorods film was rinsed and annealed at 500 °C for 60 min.Patterned fluorine-doped tin oxide (FTO)-coated glass substrate was cleaned by sonication for 20 min in detergent, acetone, 2-propanol, and ethanol, respectively. A TiOs report . A 0.7 m3NH3I) was synthesized with a reported method [2 and CH3NH3I in anhydrous N,N-dimethylformamide (DMF) at a 1:3 molar ratio at 60 °C overnight.Methylammonium iodide  curves were carried out with a Keithley 2440 Source meter under AM 1.5 G illumination from a Newport Oriel Solar Simulator with an intensity of 100 mW/cm2. A shadow mask was used to determine the active area of 0.1 cm2. Morphologies and microstructures were performed using a scanning electron microscope  equipped with an energy dispersive spectrometer (EDS). Absorption spectra were obtained with a UV–Vis spectrophotometer (Varian Cary 5000). Steady-state photoluminescence (PL) and time-resolved photoluminescence (TRPL) spectra were collected with a fluorometer . An electrochemical workstation  was used to collect the electrochemical impedance spectroscopy (EIS) with a bias of 0.6 V.X-ray diffraction (XRD) patterns were measured using a diffractometer (DX-2700). Photocurrent–voltage  and forward scan (FS). The photovoltaic parameters were obtained by the average of reverse scan and forward scan for each device. The Isc, Voc, FF, and PCE of the solar cells in the study were obtained by an average of the data from 20 pieces of devices. Figure 2 NRs. The Isc, Voc, FF, and PCE of the solar cells based on N-TiO2 NRs are enhanced compared with those based on un-doped TiO2 NRs. The PCE of solar cells based on N-TiO2 NRs is 14.7% higher than that of solar cells based on un-doped TiO2 NRs. Figure I–V curves of best performance solar cells based on un-doped TiO2 and 1% N-TiO2 NRs.TiO2 NRs and 1% N-TiO2 NRs. The IPCE at the entire wavelength range of the cells based on N-TiO2 NRs are larger than those on the un-doped TiO2 NRs, which agree with the I–V measurements.Figure 2 and 1% N-TiO2 NRs. The peaks labeled with stars were assigned to SnO2  on FTO substrate. The peak at 36.1° was assigned to the (101) planes of rutile TiO2  [2 NRs, the peaks on N element were not observed. This could be due to the homogeneous distribution of N with Ti in the samples [To get an insight into the enhancement, some investigations were performed. Figure  samples  and smal2 NRs. Figure 3/2 and Ti 2p1/2, respectively. The peak located at 400.1 eV in Fig. 2 film, which was further confirmed by the EDS spectrum as shown in Additional file Figure 2 NRs, and Fig. 2 NRs. The diameter and length of the un-doped TiO2 NRs were determined to be 48 ± 5 nm and 490 ± 15 nm, respectively. The diameter and length of the 1% N-TiO2 NRs were determined to be 42 ± 6 nm and 480 ± 25 nm, respectively. The diameter of N-TiO2 NRs is slightly decreased compared with that of un-doped TiO2 NRs. The length distribution of N-TiO2 NRs is more ununiform than that of un-doped TiO2 NRs. From a view of large area  can be calculated based on the absorption spectra using the Tauc equation [2 and N-TiO2 NRs, respectively. Compared with that of un-doped TiO2, the energy band gap of N-TiO2 becomes smaller. This could be attributed to the substitution location of N in the TiO2 lattice, in which an O (Ti) atom is replaced by an N atom [2 and MAPbIx3−Clx is larger than that between un-doped TiO2 and MAPbIx3−Clx due to its narrow energy band gap, which might be one of the reasons to present a higher voltage for N-TiO2-based solar cells [Figure equation . The Taun N atom . The conar cells .Fig. 6a2/MAPbIx3−Clx and 1% N-TiO2/MAPbIx3−Clx. The intensity of PL spectrum was decreased due to the electron transfer when the MAPbIx3−Clx film contacts with TiO2 nanorod layer. The intensity decrease for N-TiO2 is more significant than that for un-doped TiO2, which indicates the electron transfer from MAPbIx3−Clx layer to N-TiO2 is more effective than to un-doped TiO2.PL is a suitable tool to study the efficiency of charge carrier trapping, migration, and transfer , 28. Fig2/MAPbIx3−Clx and 1% N-TiO2/MAPbIx3−Clx. The TRPL curves were analyzed with a bi-exponential decay function containing a fast decay (τ1) component and a slow decay component (τ2), and the detailed parameters are listed in Table x3−Clx to TiO2, and the slow decay component could be the result of radiative decay [2/MAPbIx3−Clx, the fast decay time is 55.1 ns, and the slow decay time is 121.4 ns, while their weight fractions are 31.3 and 67.7%, respectively. Compared with those of un-doped TiO2/MAPbIx3−Clx, the fast decay lifetime of N-TiO2/MAPbIx3−Clx is decreased to 36.4 from 55.1 ns, and the slow decay life time to 109.5 from 121.4 ns, while the weight fraction of fast decay is increased to 35.1 from 31.3%. This suggests that N-TiO2/MAPbIx3−Clx interface presents a faster charge transfer and induced charge recombination than the un-doped TiO2/MAPbIx3−Clx interface. The performance difference between the solar cells based on un-doped TiO2 and N-doped TiO2 NRs could be due to the property change of ETM, which affects the charge behavior in the interfaces [Figure ve decay , 30. In terfaces .Table 2P2 and 1% N-TiO2 NRs. The EIS contains two RC arcs. The arc at high frequency is originated from the contact resistance of the interfaces and that at low frequency is attributed to recombination resistance (Rrec) and chemical capacitance (Cμ) of the device [2 present smaller series resistance and larger recombination resistance than those on un-doped TiO2. This demonstrates that the charge transport ability was enhanced and the carrier recombination rate was induced for the device on N-TiO2. There are many surface and bulk trap states due to oxygen vacancies for TiO2. It demonstrated that the incorporation of N atom into the lattice of TiO2 can decrease the traps density probably due to oxygen vacancy reduction [2 NRs could be due to the decreased traps density. The larger recombination resistance of N-TiO2 NRs may be contributed to the inactive N element leading to the increase of surface resistance [To understand the charge transfer behavior of the solar cells, electrochemical impedance spectrum (EIS) was measured. Figure e device , 34. Figeduction , 35. Thesistance , 37.Fig.2 NRs were synthesized with hydrothermal method, and perovskite solar cells based on them were fabricated. Compared with the solar cells based on un-doped TiO2, solar cells based on N-TiO2 present an enhanced performance. The solar cell performance was optimized by changing the N doping contents. The PCE of solar cells based on N-TiO2 with the N doping content of 1%  has been achieved 11.1%, which was 14.7% higher than that of un-doped TiO2-based solar cells. To explain this phenomenon, some investigations were performed. The results indicate that the larger Voc could be due to the larger conduction band offset resulting from the smaller energy band gap for N-TiO2, and the enlarged Isc could be attributed to the faster electron transfer and reduced recombination rate for N-TiO2 NRs. These induce the enhanced performance of the solar cells based on N-TiO2 NRs.In the present study, N-TiOAdditional file 1:Figure S1. EDS spectrum of 1% N-TiO2 NRs. Figure S2. Plane-view SEM images of un-doped TiO2 (A), and 1% N-TiO2 NRs(B). Figure S3. Cross-sectional SEM image of perovskite solar cells. (DOCX 826 kb)Supplementary Material.