Vitamin D is important to maternal, fetal, and infant health, but quality data on vitamin D status in low- and middle-income countries and response to cholecalciferol supplementation in pregnancy are sparse.We characterized vitamin D status and vitamin D metabolite change across pregnancy and in response to cholecalciferol supplementation in rural Gambia.3], 24,25-dihydroxycholecalciferol , and C3-epimer-25-hydroxycholecalciferol [3-epi-25(OH)D3] were measured by LC-MS/MS in 863 women [aged 30 ± 7 y (mean ± SD)] in early pregnancy (presupplementation) and late pregnancy, . Changes in 25(OH)D3 and vitamin D metabolite concentrations and associations with pregnancy stage and maternal age and anthropometry were tested.This study was a secondary analysis of samples collected in a 4-arm trial of maternal nutritional supplementation ; MMN included 10 μg/d cholecalciferol. Plasma 25-hydroxycholecalciferol [25(OH)D3 concentration was 70 ± 15 nmol/L and increased according to pregnancy stage (82 ± 18 and 87 ± 17 nmol/L in the FeFol and PE-arms) and to cholecalciferol supplementation (95 ± 19 and 90 ± 20 nmol/L in the MMN and PE + MMN-arms) (P < 0.0001). There was no difference between supplemented groups. Early pregnancy 25(OH)D3 was positively associated with maternal age and gestational age. Change in 25(OH)D3 was negatively associated with late pregnancy, but not early pregnancy, triceps skinfold thickness. The pattern of change of 24,25(OH)2D3 mirrored that of 25(OH)D3 and appeared to flatten as pregnancy progressed, whereas 3-epi-25(OH)D3 concentration increased across pregnancy.Early pregnancy 25(OH)DThis study provides important data on the vitamin D status of a large cohort of healthy pregnant women in rural Africa. Without supplementation, vitamin D status increased during pregnancy, demonstrating that pregnancy stage should be considered when assessing vitamin D status. Nutritionally relevant cholecalciferol supplementation further increased vitamin D status. These data are relevant to the development of fortification and supplementation policies in pregnant women in West Africa. Vitamin D is of relevance to maternal, fetal, and infant health globally in relation to pregnancy-related complications , preterm birth, and infant-related outcomes , 2. In pGlobally, 54% of pregnant women are estimated to have a plasma/serum total 25-hydroxyvitamin [25(OH)D] concentration <50 nmol/L and 18% <25 nmol/L indicating a concerning degree of vitamin D insufficiency and deficiency . More reChanges in 25(OH)D concentration, the primary marker of vitamin D status, are not well characterized throughout pregnancy and data are conflicting . Lack of2D] may provide information on the relative activation of catabolic pathways D, are not well defined , 25. Chapathways , 27, incpathways . ChangesD status , 29. In D status .Vitamin D supplementation is generally effective in raising plasma/serum 25(OH)D concentrations, including in pregnant women . However1) characterize vitamin D status in a large pregnancy cohort in sub-Saharan Africa, 2) determine the impact on vitamin D status of nutritionally relevant daily doses of supplemental vitamin D (10 μg/d), and 3) describe vitamin D metabolite concentrations across gestation.Therefore, in a secondary analysis of samples collected as part of a randomized controlled trial of multiple-micronutrient supplementation including vitamin D, and designed to enhance infant immune development , 35, 25(Samples and data were collected as part of the ENID (Early Nutrition and Infant Immune Development) trial (ISRCTN49285450) conducted from MRC Keneba, MRC Unit The Gambia in the rural area of West Kiang, a primarily subsistence farming community. At a latitude of 13°North, UVB-containing sunshine is available year-round , and mosFull details of the ENID trial have been published , 35, 39 3). A full description of the contents of each supplement can be found in Moore et al.  testing  and then confirmed and gestational age assessed by ultrasound . After confirmation of pregnancy, women were randomly assigned to 1 of the 4 intervention arms, iron-folic acid (FeFol) tablets, multiple micronutrient (MMN) tablets, a protein-energy (PE) lipid-nutrient supplement (LNS), or the PE supplement with multiple micronutrients (PE + MMN). FeFol is the standard supplement advised in pregnancy according to Gambian Government guidelines. MMN arms included 10 μg/d (400 IU) of cholecalciferol D3], and C3-epimer of 25-hydroxycholecalciferol [3-epi-25(OH)D3] was performed based on a published LC-MS/MS method . The limit of quantification (LOQ) was 1.5 nmol/L for 25(OH)D2, 25(OH)D3, and 3-epi-25(OH)D3 analytes, and 2.5 nmol/L for 24,25(OH)2D3. The MRC Elsie Widdowson Laboratory is a member of the Vitamin D Standardization Program, and quality assurance of the assay was performed as part of the Vitamin D External Quality Assessment Scheme (www.deqas.org) and performance assessed against NIST SRM 972a .The quantitation of plasma 25-hydroxyergocalciferol  are presented as geometric mean and geometric mean SD. Parity is presented as median and range.Data analysis was performed with Stata 14.2 (StataCorp LLC). Because only a few samples contained 25(OH)Doot of 2 ; assigneSupplement group differences in both early and late pregnancy, and differences between early and late pregnancy were tested by ANOVA. Pairwise comparison of group means was tested post hoc with Scheffé procedure. For skewed data, analysis was performed using logged values. Pearson's chi-square test was used for categorical data and the Kruskal-Wallis H to test for equality of medians . Tests were not adjusted for weeks of pregnancy because there was no difference between supplement groups in this parameter.3 concentration was investigated with the use of Fourier regression Dgression , 43, witariation . In mult3 concentration with use of linear regression. Significant predictors of age and weeks of pregnancy were included in the multivariate model, which also included Fourier terms to control for any effect of time of year. To investigate predictors of attained 25(OH)D3, both early pregnancy 25(OH)D3 concentration and supplementation group were included in all models.We investigated biologically plausible predictors of early pregnancy 25(OH)D2D3 and % 3-epi-25(OH)D3 refer to the relative concentration of the metabolite to 25(OH)D3 concentration, that is 24,25(OH)2D3/25(OH)D3 *100.Vitamin D metabolite concentrations and their ratios were compared between pregnancy stage and supplementation group using ANOVA with Scheffé post hoc test. Percent 24,25(OH)P > 0.05). Metabolite concentrations below the LOQ were excluded for this and for the following analysis; results with these data are included in the Supplemental Table 2).The effect of gestational age on vitamin D metabolite concentrations and the ratios of the metabolites to 25(OH)D were investigated using a mixed linear model including weeks of pregnancy (fixed effect) and participant ID (random effect). To describe the normal physiological pattern in pregnancy, this analysis was performed in the FeFol group only. Possible nonlinear associations were assessed by the inclusion of a quadratic term (predictor*predictor), but removed from the model if not significant . Data from the 4 groups were pooled because there were no supplement group differences in 25(OH)D and metabolite relations or ratios (investigated by the inclusion of an interaction term between supplement group and the predictor variable). Differences between early and late pregnancy in metabolite relations were tested with the inclusion of interaction term between time point (early/late pregnancy) and the continuous predictor variable.Relations between vitamin D metabolites and their ratios against 25(OH)D3 and vitamin D metabolite concentrations are shown in P > 0.5). In early pregnancy, the gestational age ranged between 7.0 and 20.8 wk. The distribution of the month of sampling was not different between supplementation groups (P = 1.0).Participant characteristics and early pregnancy plasma 25(OH)D3 concentration was 70.2 ± 15.3 nmol/L. No woman had 25(OH)D3 <30 nmol/L. 25(OH)D2 was present above the LOQ in 50 (6%) samples in early pregnancy and in these women the mean concentration was 1.8 ± 0.4 nmol/L, consistent with low availability of vitamin D2 in this population.In early pregnancy, the mean plasma 25(OH)D3 concentration clearly visible in the vitamin D-supplemented groups  D3 concentration between early and late pregnancy. Vitamin D supplementation shifted the cohort distribution, reducing the percentage of participants in the MMN group with 25(OH)D3 concentration <50 nmol/L to zero, but increasing the proportion with a concentration >125 nmol/L D groups) . In the 5 nmol/L ; Table 13 plasma concentration  . Investigated predictors of early pregnancy, presupplementation, and 25(OH)D3 concentration are shown in 3 concentration was not associated with maternal size or adiposity, but maternal age and weeks of pregnancy positively predicted 25(OH)D3 concentration D3 concentration for a 1-wk increase in gestational stage (P < 0.0001) D 0.0001) .3 concentration in late pregnancy were further investigated using a combined model with presupplementation, baseline 25(OH)D3 concentration, and study arm as covariates. In these combined models there were no significant interactions between predictor variables and supplementation group. Attained 25(OH)D3 concentration was positively associated with compliance and weeks on supplementation, and negatively with late pregnancy triceps skinfold thickness (3 concentration was 0.2 nmol/L lower (P = 0.02). There were no associations with other anthropometric indices measured in either early or late pregnancy.Participant characteristics influencing 25(OH)D3 and 24,25(OH)2D3 across gestation such that the positive linear increase observed in early pregnancy appeared to flatten towards late pregnancy. In contrast, 3-epi-25(OH)D3 concentration increased as pregnancy progressed 2D3 ratio was consistent across gestational age, whereas we observed a significant negative relation between the 25(OH)D3:3-epi-25(OH)D3 ratio and gestational age.We observed quadratic relations in both 25(OH)Dogressed , C. Thesogressed , E; the 2D3 are reported in 2D3 and 25(OH)D3 concentration that was not different between early and late pregnancy (P = 0.3 interaction) 2D3 and 25(OH)D3 (P = 0.03), but this was not significantly different from the nonsignificant relation observed in late pregnancy (P = 0.1 for interaction) (2D3 concentrations that were below the LOQ (2D3 and 25(OH)D3 were similar to those obtained using data above the LOQ. However, in contrast to using data above the LOQ, there were significant negative linear relations between the ratio 25(OH)D3:24,25(OH)2D3 with 25(OH)D3 in both early (β ± SE)  and late pregnancy .Plasma concentrations of 24,25(OH)raction) . We also the LOQ . In this3 are reported in 3 were higher than in early pregnancy across all supplementation groups (3 and both 3-epi-25(OH)D3 concentration and their ratios were not different between early and late pregnancy (P > 0.4) (Plasma concentrations of 3-epi-25(OH)Dn groups . The sloP > 0.4)  and D.3 concentration increased by 11 nmol/L across pregnancy in the group not receiving supplemental vitamin D. Weeks of pregnancy was a strong predictor of vitamin D status and should therefore be considered in the interpretation of studies of vitamin D status in pregnancy. Secondly, in groups receiving 10 μg/d (400 IU/d) vitamin D3 within a micronutrient supplement, there was an additional ∼11 nmol/L increase in 25(OH)D3 concentration across pregnancy. Thirdly, in women not receiving vitamin D supplementation, 3-epi-25(OH)D3 concentration continued to increase across pregnancy suggesting an independent effect of pregnancy on production of 3-epi-25(OH)D3.We have characterized vitamin D status, response to supplementation, and vitamin D metabolite concentrations in a large cohort of pregnant women resident in rural Gambia. This study generates important findings in relation to vitamin D physiology in pregnancy. Firstly, we observed that during pregnancy in women with relatively constant vitamin D supply from cutaneous synthesis and generally good vitamin D status, 25(OH)DThis is the largest study of vitamin D status in a pregnancy cohort in Africa. The Gambia lies relatively close to the equator at 13°N and therefore receives year round UVB-containing sunshine . AlthougVitamin D status was relatively high in this population with a mean of ∼70 nmol/L in early pregnancy; there was no vitamin D deficiency [25(OH)D concentration <30 nmol/L] , 32. Thi3 concentration in early pregnancy and indicates that this variable should be accurately determined and included in the analysis of studies of vitamin D status during pregnancy. The observed ∼11 nmol/L increase in 25(OH)D3 concentration across pregnancy is equivalent to the increase expected with an oral intake of ∼400 IU/d of vitamin D. Reported changes in vitamin D status with gestation are inconsistent, partly because of the large seasonal influence present in many studies, with suggestions of increases D is maintained, when permitted by a sufficiently high supply of vitamin D. Recent data from a study in pregnant US adolescents suggested a slight negative association between % free 25(OH)D and 25(OH)D concentration, although free 25(OH)D was highly correlated with 25(OH)D and did better predict associations with parathyroid hormone Dncreases , 46, D3 concentration with daily doses of 10 μg/d was consistent with the dose-response observed in studies in other ethnic groups, including in pregnancy (Vitamin D supplementation in this largely vitamin D-replete population had a positive effect on vitamin D status and reduced the proportion of women with both 25(OH)Dregnancy , 51. An regnancy , whereasregnancy , 53.3 concentration in cord blood of >50 nmol/L  at which the Institute of Medicine reported as a cause for concern  (Vitamin D supplementation in our study increased the proportion of women with 25(OH)Dof data) . As reviof data) , 54–56. of data) , and the3 concentration, after adjustment for other factors. The relation with skinfold thickness may only have become apparent in late pregnancy after oral vitamin D because of differences in vitamin D transport. The tissue distribution of oral vitamin D that is initially transported by chylomicrons and lipoproteins may be different to that of cutaneously synthesized vitamin D transported by DBP D3, increased with rising 25(OH)D3 concentration. For 24,25(OH)2D3, the increase was followed by a flattening that closely mirrored that of 25(OH)D3. In contrast, 3-epi-25(OH)D3 concentration continued to rise, an observation that was reflected when its ratio was plotted against weeks of pregnancy. This is consistent with recent work that reported an increase in 3-epi-25(OH)D3 as pregnancy progressed D3 results are similar to those estimated from the results from some studies D3 concentrations (<∼25 nmol/L) D3 concentration (2D3 may be more related to 25(OH)D3 because the ratio across gestation did not change. The 24,25(OH)2D3:25(OH)D3 ratio is lower than reported in studies of nonpregnant adults ogressed . As a pe studies . The souneonates , are unkneonates . It is s nmol/L) , and Best adults , but con2, 25(OH)D3, 3-epi-25(OH)D3, and 24,25(OH)2D3 provides confidence over the change in vitamin D status over pregnancy as well as providing data on vitamin D metabolite changes over pregnancy.An advantage of this study was our ability to determine changes in vitamin D status across pregnancy in a relatively large number of individuals where the effect of season was small. In contrast to some studies, the use of an LC-MS/MS method that fully resolved and quantified 25(OH)D2D3 was higher than in some other studies D and vitamin D metabolite response to pregnancy and vitamin D supplementation when vitamin D supply is stable from year-round high UVB availability. The generalizability of these findings to other populations requires further study in different groups. The potential relevance of nutritionally relevant doses (10 µg/d) and their impact in vitamin D supplementation trials and relevance to the development of vitamin D fortification and supplementation policies in pregnant women are highlighted.nxz290_Supplemental_FileClick here for additional data file.


Allium cepa Linn.). The aims of the present study were to isolate and identify PGPB in onion and examine the effects of isolated PGPB on germination and growth during the vegetative stage in onion, pak choy (Brassica chinensis), and sweet pepper (Capsicum annuum). Twenty-three strains of PGPB were isolated from the roots, bulbs, and rhizosphere soil of onion. All isolated bacterial strains showed one or more PGP traits, including indole acetic acid production, phosphate solubilization ability, and 1-aminocyclopropane-1-carboxylate deaminase and nitrogenase activities; most of these traits were derived from Bacillus sp., Microbacterium sp., and Pseudomonas sp. Eight bacteria that exhibited strong abilities to produce indole acetic acid were selected for a Petri dish trial, soil pot test, and vermiculate pot test. The Petri dish trial showed that strains ORE8 and ORTB2 simultaneously increased radicle and hypocotyl lengths in onion, but inhibited growth in sweet pepper after 7 d. The soil pot experiment on onion revealed that strains ORE5, ORE8, and ORTB2 strongly promoted growth during the vegetative stage with only a half dose of chemical fertilizer. The present results indicate that ORE8 (Bacillus megaterium) and ORTB2 (Pantoea sp.) are the most promising biofertilizers of onion and may simultaneously inhibit the seedling growth of other plants.Plant growth-promoting bacteria (PGPB) are beneficial microbes that increase plant growth and yield. However, limited information is currently available on PGPB in onion ( Modern crop production involves protecting the environment, maintaining soil fertility, and controlling pests effectively while achieving reasonable yield. This task is more challenging in Taiwan, located in a subtropical region, in which small and fragmented cultivated lands and high temperature and humidity all contribute to serious competition between pests and crops. The use of chemical fertilizers and pesticides has always been the main approach to promote agricultural production in Taiwan. However, the long-term use of chemical fertilizers and pesticides has affected the original environment, deteriorated soil fertility, generated highly resistant pests, and raised public concerns about the environment and health.Allium cepa Linn.)  have attracted increasing attention because they promote the growth of crops such as maize, rice, and sugarcane (nn.) . The seedling growth of radish was previously shown to be suppressed by high IAA-producing s of IAA . Auxin hs of IAA .Pseudomonas fluorescence and Bradyrhizobium elkanii  were collected from a farmer’s plantation in Taichung County, Taiwan. To isolate onion root endophytes, the roots were rinsed with distilled water to remove debris and then sterilized with 75% alcohol for 15 min, followed by immersion in 6% sodium hypochlorite for another 15 min. Roots were washed with sterilized water after the sterilization process. Samples were ground as described previously  plates. The same procedure was used to isolate strains from bulbs. However, the method of Onion plants . Bacterial strains were inserted into the middle of the medium plates using sterilized tips. Plates were incubated at 30°C for 7 d to monitor the positive or negative development of a clear halo around the colonies.Bacterial isolates were screened for phosphate solubilization properties using Pikovskaya medium  and incubated at 30°C for 24 h on a rotating shaker (180 rpm). A 20-μL amount of the culture was transferred into 5-mL test tubes containing NB with 500 μg mLthe dark .g at 4°C for 10 min. Bacteria cell pellets were collected and washed with 1 mL of 0.1 M Tris-HCl (pH 7.6) at 4°C and washed cell pellets were re-suspended in 600 μL of 0.1 M Tris-HCl (pH 8.5). A 30-μL amount of toluene was added to the cell suspension and vortexed for 30 s. The enzymatic reaction was initiated by adding 20 μL ACC to 200 μL of toluenized cells after a pre-incubation at 30°C for 3 min. After a 15-min reaction, 1 mL of 0.56 M HCl was added. The mixture was collected and centrifuged at 10,000×g at room temperature for 5 min. A 1-mL amount of the supernatant with 800 μL of 0.56 M HCl was vortexed. A 300-mL amount of 2,4-dinitrophenylhydrazine reagent was added and incubated at 30°C for 30 min. Two milliliters of 2 N NaOH was added to each test tube and absorbance was recorded at 540 nm. The known concentration range of 0 to 1.0 mmol α-ketobutyrate was used as a standard.Experiments to measure ACC deaminase in strains were conducted as described previously . Each ba–1; temperature of the flame ionization detector, 110°C; column (1 m×2 mm steel column packed with a Porapak-T 80–100 mesh); temperature, 80°C.Nitrogen fixation was evaluated using the acetylene reduction assay . After g5′-AGAGTTTGATCCTGGCTCAG-3′; 1492R 5′-GGTTACCTTGTTACGACTT-3′ was used to amplify a 1,500-bp region. PCR amplification involved a 50-μL reaction mixture consisting of 50 ng genomic DNA, 20 pmol of each primer, 1.25 units Taq DNA polymerase, 200 μM of each dNTP, and 1× PCR buffer. PCR was performed for 32 cycles in a Mycycler (Bio-Rad) with initial denaturation at 95°C for 3 min, cyclic denaturation at 94°C for 30 s, annealing at 58°C for 30 s, and extension at 72°C for 2 min with a final extension of 7 min at 72°C. The amplicon of each reaction was analyzed by 1% agarose gel staining with ethidium bromide and visualized under UV light with a gel documentation system. Amplicons were sent to Genomics BioSci & Tech for sequencing.Bacterial isolates were identified by targeting 16S rDNA . The prihttp://www.eztaxon.org). The 16S rRNA gene sequences obtained were analyzed using the sequence alignment Blast program from NCBI (https://blast.ncbi.nlm.nih.gov/Blast.cgi) and then aligned with the sequences of closely related strains using CLUSTAL_X 1.83 . Closely related type strains were obtained from NCBI GenBank via the EzTaxon server (L_X 1.83 . A bootsL_X 1.83  was perfL_X 1.83  based onL_X 1.83 .Brassica chinensis), and sweet pepper (Capsicum annuum)—to examine their effects on the germination of seeds and elongation of radicle and hypocotyl lengths. Strains were grown on NA plates for 3 d and then suspended in RO water. A 2-mL amount of the cell suspension at A600=0.1 was added to filter paper (AdvantecToyo) in a Petri dish. Seeds were surface-sterilized in 1% sodium hypochlorite for 15 min. Plates were then incubated at 30°C for 7 d. We used five replicates for each strain. The hypocotyl and radicle lengths of three crops were measured after 7 d of culture.Among the 23 strains isolated from the onion plant parts, 8 showing high IAA production abilities were introduced into three vegetable crops—onion, pak choy  was collected from an onion farm in Taichung county. To inoculate bacteria on seeds, 5-mL cell suspensions from 3-days-old colonies were added on filter paper to Petri dishes. Each treatment was replicated four times. The treatments included the following: half-dose fertilizer control (1/2F), half-dose fertilizer (1/2F)+individual strain, 1/2F+mixture of the 5 strains, full-dose fertilizer control (1F), 1F+individual strain, and 1F+mixture of the 5 strains. Four replicates for each strain were used (600=0.1) from 3-days-old bacterial cells, incubated at 25°C for 7 d, and then transplanted to pots with unsterilized soil (sieved and air-dried soil). A 5-mL amount of the cell suspension in sterile water was added to pots immediately after transplanting and 1 week after transplanting. Plants were grown in a growth chamber for 7 weeks with a 12-h photoperiod (light: 153–179 μmole photons s–1 m–1) at 25°C. Pots were watered regularly and uniformly  containing 0.5 kg air-dried and sieved (2-mm mesh) soil. Soil  pots. To simplify the growth environment, we used vermiculite and not the soil sampled from the onion field.Before planting, vermiculite was autoclaved once every day for 2 consecutive days. Unless indicated, bacteria preparation and inoculation, seed treatments, and pot sizes were all described previously . We used four replicates for each strain.–1. Plant height and root length were measured after 4 weeks.The treatments included a control with half fertilizer (1/2F) and 1/2F+each of the 8 strains. Seeds were directly sown in vermiculite pots, and grown in a greenhouse for 4 weeks at National Chung Hsing University, Taiwan. A 5-mL amount of the cell suspension in sterile water was added to pots when sown and 1 week after sowing. Pots were watered regularly and uniformly  with a one-way ANOVA and post-hoc test with the LSD test. Microbacterium hydrocarbonoxydans, ORLB2 was 98.6% identical with M. laevaniformans, and ORTB2 was 97.6% identical with Pantoea sp.. BLAST results were further confirmed with a phylogenetic tree .Twenty-three strains with PGP traits were isolated and identified from the bulbs, rhizosphere, and roots of onion plants . The 16Stic tree . Five stB. cereus isolated from the roots showed negative results. After 7 d, some strains were able to solubilize more phosphorus than other strains based on the size of the transparent circle (data not shown). ORE1, ORE5, ORE8, and ORE10 isolated from onion roots showed larger transparent circles than other strains  and Pantoea sp. (ORTB2) produced the largest amounts of IAA at 527 and 462 mg mL–1, respectively. The strains that produced the most IAA were isolated from the root endophyte (~103.8 mg mL–1 on average) and rhizosphere soil (~111.8 mg mL–1 on average), but not from the bulb (~6.8 mg mL–1 on average).As shown in –1 ΔOD600–1). The strains that produced the strongest ACC deaminase activity were isolated from the rhizosphere, with only one strain being isolated from the onion bulb (OBE2). The remaining strains exhibited between 97- and 500-nmol α-ketobutyrate h–1 ΔOD600–1 ACC deaminase activity.All isolated strains were positive for ACC deaminase activity . Some st2H4 h–1, with OBE4 (25.9 nmol C2H4 h–1) exhibiting the strongest nitrogenase activity and ORE7 (2.9 nmol C2H4 h–1) the weakest activity. Among the potential biofertilizers selected in the present study for subsequent plant experiments, ORTB3 had the highest nitrogen-fixing ability (8.1 nmol C2H4 h–1). Three out of the four bacterial strains with the highest abilities were from the onion bulb (~12.7 nmol C2H4 h–1 on average). The strains with the lowest ability were from the root endophyte (4.9 nmol C2H4 h–1 on average), followed by those from the rhizosphere (6.0 nmol C2H4 h–1).All bacterial strains in semi-solid nitrogen-free medium were positive for acetylene reduction (or nitrogen-fixing) activity when analyzed by gas chromatography . The ranWe selected eight out of 23 strains for the Petri dish trial because of their high production levels of IAA , G. terrae (ORE9), and K. oxytoca (OBE4) have been isolated from human clinical specimens or human pathogens (B. megaterium (ORE8) and Pantoea sp. (ORTB2) were selected because of their high IAA yield.However, in addition to their plant and soil origins, B. megaterium strains isolated from root endophytes , Leifsonia sp. (ORE5) also isolated from root endophytes, and M. laevaniformans (ORLB2), P. fluorescens (ORTB3) and P. moraviensis (ORTB5) isolated from roots. B. megaterium and P. moraviensis were previously shown to produce a large amount of IAA  and ORTB2 (Pantoea sp.) inhibited the growth of sweet pepper, ORTB2 the growth of pak choy, and both strains the growth of onion, possibly because these two bacteria had the highest IAA production abilities among the bacteria tested  and phosphate-solubilizing abilities.Based on the results of our Petri dish trial , ORE8  perceive or respond differently to exogenous auxins than dicots due to the limited translocation or rapid degradation of exogenous auxins in monocots  (–1 h–1 of ACC deaminase activity was sufficient to permit a bacterium to grow on ACC and function as a PGPB. High activity levels of ACC deaminase (300 to 400 nmol α-ketobutyrate mg protein–1 h–1) for bacteria do not necessarily promote root elongation to any greater extent. However, the possibility of a dual effect from high ACC deaminase activity has not yet been reported.ACC deaminase, an enzyme that reduces ethylene levels in plants, is recognized as a PGP trait (M. laevaniformans) and ORTB2 (Pantoea sp.) at 975.0 and 909.8 nmol h–1 ΔOD600–1, respectively. However, we did not observe the similar stimulation of pak choy seedling growth by the two stains  had the highest nitrogen-fixing ability (8.1 nmol C2H4 h–1). However, in the Petri dish trial , ORE8 (B. megaterium), and ORTB2 (Pantoea sp.) promoted onion growth the most with half-dose fertilizer, while similar results were obtained for ORE5 (Leifsonia sp.), ORTB2 (Pantoea sp.), and ORTB3 (P. fluorescens) with full-dose fertilizer.In the Petri dish trial and soil pot experiment, we verified the stimulatory effects of the selected PGPB on onion growth. In the soil pot experiment, ORE5  . Strains ORE5 (Leifsonia sp.), ORE8 (B. megaterium), and ORTB2 (Pantoea sp.) induced the greatest increases in onion growth during the vegetative stage with the application of half-dose chemical fertilizer. Furthermore, ORE8 (B. megaterium) and ORTB2 (Pantoea sp.) were the most promising biofertilizers for onion, but may simultaneously inhibit the seedling growth of other plants. Nevertheless, future field studies for the above mentioned bacteria need to be conducted in order to verify the effect on onion and the targeted plant species.In conclusion, we isolated 23 bacterial strains from an onion plant. Most of the bacterial strains were Allium cepa Linn.. Microbes Environ 35: ME19147.Samayoa, B. E., Shen, F.-T., Lai, W.-A., and Chen, W.-C. (2020) Screening and Assessment of Potential Plant Growth-promoting Bacteria Associated with https://doi.org/10.1264/jsme2.ME19147


Urolithin A also acted as a direct radical scavenger, showing values of 13.2 μM Trolox Equivalents for Oxygen Radical Absorbance Capacity (ORAC) and 5.01 µM and 152.66 µM IC50 values for superoxide and 2,2-diphenyss1-picrylhydrazyl (DPPH) radicals, respectively. Finally, inhibition of oxidizing enzymes, such as monoamine oxidase A and tyrosinase, was also detected in a dose-dependent manner. The cytoprotective effects of urolithin A could be attributed to the improvement of the cellular antioxidant battery, but also to its role as a direct radical scavenger and enzyme inhibitor of oxidases.Urolithin A is a metabolite generated from ellagic acid and ellagitannins by the intestinal microbiota after consumption of fruits such as pomegranates or strawberries. The objective of this study was to determine the cytoprotective capacity of this polyphenol in Neuro-2a cells subjected to oxidative stress, as well as its direct radical scavenging activity and properties as an inhibitor of oxidases. Cells treated with this compound and H Melaleuca quinquenervia leaves   × 100The inhibition of AChE was carried out in 96-well microplates, measuring the absorbance at 450 nm 11 times using a Synergy H1 Hybrid Multi-Mode Reader . Each weThe MAO-A inhibition assay was performed following a protocol described by Olsen et al. . Here, 5The capacity or urolithin A to scavenge peroxyl radicals was measured by the oxygen radical antioxidant capacity (ORAC). Trolox was used as a reference standard for this assay. Therefore, data were represented as μmol Trolox equivalents (TE)/mg sample. Different concentrations of urolithin A (4.4 μM–4.4 mM) were dissolved in PBS and methanol (50:50) and placed into the wells. Urolithin A and Trolox were incubated with fluorescein (70 mM) in 96-well plates at 37 °C. Finally, AAPH (12 mM) was added and fluorescence was measured every 70 s for 1 h and 33 min at 485 nm (excitation) and 520 nm (emission), in a Synergy H1 Hybrid Multi-Mode Reader  .2CO3 were mixed in phosphate buffer (pH = 6.9). Then, 240 µL of this cocktail was added to the well. Next, 30 µL of urolithin A and 30 µL of xanthine oxidase (168 U/L) were added to start the reaction. Before measurement, the plate was incubated for 2 min at 37 °C. The superoxide radicals’ (O2−) scavenging activity was assessed spectrophotometrically at 560 nm. The inhibitory activity of XO was also assayed spectrophotometrically at 295 nm. Gallic acid was used as a reference antioxidant compound. Another antioxidant test was performed in order to evaluate the antioxidant activity of urolithin A using a more physiological system of superoxide radical generation . Here, 2Here, 2,2-diphenyl-1-picrylhydrazyl (DPPH) is a purple free radical. Antioxidant reducing compounds are able to scavenge these free radicals, inducing a change in the color of this compound. A methanolic stock solution of DPPH (0.11 mM) was prepared and 150 µL was added together with 150 µL of different urolithin A concentrations dissolved in MeOH. Ascorbic acid and gallic acid were also measured as antioxidant standards. Control wells contained 150 µL of MeOH instead of urolithin A. The plate was incubated for 30 min under dark conditions and measured at 517 nm . Radical50 values of urolithin A and reference compounds using Student’s t-test.Each experiment was performed at least three times on different days and results were expressed as the mean ± standard error (SE) of different assays. GraphPad Prism v.6  was required to perform data analyses, nonlinear regressions, and statistics (such as two-way ANOVA or one-way ANOVA). Statistical differences were detected by comparing ICThe viability of Neuro-2a cells was evaluated by the MTT assay. In this case, different physiological concentrations of urolithin (0.5–50 µM) were tested in neurons for 24 h A. Result2O2) and exposure times  determined that incubation of hydrogen peroxide for 45 min at 250 µM was the most appropriate time period for inducing oxidative stress in N2a cells. The next purpose was to evaluate the protective effects of urolithin A on Neuro-2a cells using hydrogen peroxide as a neurotoxic insult. Different conditions (100 µM to 1000 µM of Hp < 0.005) and the metabolite .Results obtained after three replications in three different lysates showed a dose-dependent tendency of catalase for the treatments with urolithin A A. Surprip < 0.05).Different outcomes were found for the superoxide dismutase (SOD) enzyme B; a dosep < 0.01; p < 0.005). However, significant differences in GPx activity were only found at 2 µM urolithin A (p < 0.05). Comparable effects were obtained for GR and GPx activities C,D. Actip < 0.05; p < 0.01).50 values were calculated by non-linear regression for kojic acid, clorgyline, and galantamine reference inhibitors, respectively.As described above, the potential neuroprotective activity of urolithin A was tested on different enzymes present in the central nervous system . IC50 values were 24.39 ± 5.97 µM and 71.44 ± 10.07 µM for kojic acid and urolithin A, respectively). 50 value is relatively lower than urolithin A . Again, it is seen how the metabolite draws a dose-dependent curve.Finally, urolithin A was not considered as an inhibitor of AChE because very high non-physiological concentrations (876 µM) were used to reach 50% of inhibition.50 values were calculated  acid were also tested and IClculated .The antioxidant activity measured by the ORAC assay was 13.1 µmol TE/mg for urolithin A; this result reflects a great antioxidant capacity to neutralize peroxyl radicals compared to other high-content urolithin A extracts, such as pomegranate, the activity of which was 0.49 µmol TE/mg.50 values were 0.26 ± 0.21 µM for gallic acid and 5.01 ± 5.01 µM for urolithin A , while they were 3.10 ± 3.11 µM for gallic acid, 14.81 ± 14.90 µM for ascorbic acid, and 152.66 ± 35.01 µM for urolithin A .On the other hand, both in the inhibition of superoxide radicals and DPPH, urolithin A showed a dose-dependent curve reaching the maximum inhibition (100%). The reference compounds achieved better activity in terms of potency. ICMost of the studies involving natural products are performed with the original compounds found in the plant or food matrix, and very few are carried out with the metabolites obtained after the biotransformation in the body. Since ellagitannins are poorly absorbed in the gastrointestinal tract, urolithins have been proposed as the bioactive metabolites responsible for the beneficial effects of pomegranates and other plants containing ellagitannins. Pomegranate juice and extracts have exhibited neuroprotective properties, so here we investigate whether urolithin A, one of the major intestinal metabolites of ellagic acid, might act as a neurotherapeutic and antioxidant agent.Neuro-2a is a neuroblastoma cell line derived from mice, which has been used because of its ability to produce microtubular proteins. Few works have been done with urolithins using this cell line, but it has been demonstrated that urolithin A protects against ischemic neuronal injury by activating autophagy . This reThe antioxidant activity of urolithin A has already been established in different cell lines using in vitro procedures ,31,32. H2O2 have demonstrated that cells significantly increased SOD activity compared with cells subjected to oxidative stress with H2O2 [Frequently, when the ROS and MDA levels decrease, the activity of the cytosolic enzymes  increases; previous experiments in HepG2 cells treated with urolithin A and Hith H2O2 ; in factith H2O2 . Computaith H2O2 ; howeverith H2O2 ; neverthThis is also the first time that glutathione reductase and peroxidase activities were quantified in neuronal cells subjected to urolithin A treatments. In order to assess this pro-oxidant or antioxidant relationship of urolithin A, other researchers evaluated the in vitro antioxidant activity of the metabolite in HepG2 cancer cells ; unexpecC. elegans model [ROS generation is directly linked to neurodegenerative diseases, such as Alzheimer’s, Parkinson’s, or Huntington’s diseases. In this way, after demonstrating the potential of this metabolite in Neuro2-a cells, different bioassays were carried out involving CNS enzymatic targets known as acetylcholinesterase (AChE), monoamine oxidase A (MAO-A), and tyrosinase; these enzymes are considered pharmacological targets whose inhibition may lead to neuroprotective effects. Urolithin A acted as an inhibitor of oxidase enzymes such as MAO-A and tyrosinase, thus preventing oxidative damage of certain tissues. A human study in older adults with mild memory complaints suggests that 8 ounces of pomegranate juice taken daily over one-month improves verbal memory and alters neural activity during a visual source memory task . Synthetns model . In addins model . Recentlns model . Tyrosinns model ; howeverUrolithin A, a gastrointestinal metabolite from ellagitannins, is a promising therapeutic antioxidant agent with potential pharmaceutical or food applications for the prevention of oxidative stress-associated disorders, probably though the improvement of the cell antioxidant capacity by increasing the expression of thiol-dependent peroxidases.


Anthracyclines (ANT) are anti-tumor agents frequently used for the treatment of various cancers. Unfortunately, their clinical success is overshadowed by the emergence of drug resistance. Metabolism by carbonyl reducing enzymes (CREs) represents a critical mechanism of ANT resistance. Here, we have explored possible interactions of CREs with olaparib, an FDA-approved targeted chemotherapeutic. Although olaparib has been demonstrated to potentiate the antiproliferative effect of ANT in experimental models, the causing mechanisms remain unclear. In our study, we demonstrated that olaparib potently inhibits the AKR1C3 reductase at clinically relevant concentrations. Furthermore, we showed that this interaction mediates the reversal of ANT resistance and thus represents a critical mechanism of the synergy between ANT and olaparib. Our observations represent valuable knowledge that could be transformed into the more effective therapy of AKR1C3-expressing tumors.Olaparib is a potent poly (ADP-ribose) polymerase inhibitor currently used in targeted therapy for treating cancer cells with BRCA mutations. Here we investigate the possible interference of olaparib with daunorubicin (Daun) metabolism, mediated by carbonyl-reducing enzymes (CREs), which play a significant role in the resistance of cancer cells to anthracyclines. Incubation experiments with the most active recombinant CREs showed that olaparib is a potent inhibitor of the aldo–keto reductase 1C3 (AKR1C3) enzyme. Subsequent inhibitory assays in the AKR1C3-overexpressing cellular model transfected human colorectal carcinoma HCT116 cells, demonstrating that olaparib significantly inhibits AKR1C3 at the intracellular level. Consequently, molecular docking studies have supported these findings and identified the possible molecular background of the interaction. Drug combination experiments in HCT116, human liver carcinoma HepG2, and leukemic KG1α cell lines showed that this observed interaction can be exploited for the synergistic enhancement of Daun’s antiproliferative effect. Finally, we showed that olaparib had no significant effect on the mRNA expression of AKR1C3 in HepG2 and KG1α cells. In conclusion, our data demonstrate that olaparib interferes with anthracycline metabolism, and suggest that this phenomenon might be utilized for combating anthracycline resistance. Cancer is one of the most significant health problems worldwide, with 10 million new cases each year . These cOne of the most critical mechanisms of ANT resistance is its reduction to the less potent C13-hydroxy metabolite  by carbonyl-reducing enzymes (CREs) ,6,7. CREOlaparib  is a U.S. Food and Drug Administration (FDA)-approved targeted agent  that actGiven these considerations, the present work aimed to characterize the interaction of olaparib with selected recombinant reductases that play a role in anthracycline reduction. Subsequent experiments have evaluated whether these interactions modulate daunorubicin resistance in AKR1C3-expressing models. In the last experimental set, we investigated the possible influence of olaparib on AKR1C3 expression, which can affect the resistance phenotype of cancer cells.First, we investigated the potential of 10 and 50 µM olaparib to inhibit selected anthracycline reductases extensively involved in daunorubicin metabolism ,33. Olap50) = 2.48 µM; Ki) = 3.35 µM; α > 1; In subsequent studies with recombinant AKR1C3, we characterized its interaction with olaparib in detail. Olaparib exhibited high inhibitory affinity toward AKR1C3  were used as targets for molecular docking. As already described in the literature, residues Trp-227, Phe-306, and Phe-311 exhibit significant side-chain flexibility depending on the ligand . Thus, dTo evaluate the inhibitory effect of olaparib on daunorubicin (Daun) reduction at the cellular level, an inhibitory assay in AKR1C3 and empty vector-transfected HCT116 cells (HCT116–AKR1C3 and HCT116–EV (empty vector), respectively) was employed. A clinically relevant Daun concentration (1 µM) was used in these experiments .50 = 5.91 µM; In HCT116–AKR1C3 cells, olaparib showed significant dose-dependent inhibition of AKR1C3-mediated Daun metabolism . In accordance with the recognized role of AKR1C3 in Daun resistance, the AKR1C3-overexpressing subline showed reduced sensitivity to Daun compared to the EV-transduced subline . Olaparib potentiated the effect of Daun in both HCT116 sublines, with a trend toward increased effects in the AKR1C3-overexpressing cell line (A)  c. At 10 line (A) d. To excolaparib ,39) exprA in KG1α leukemic cells  analysis to assess possible changes in AKR1C3 expression in either HepG2 or KG1α cells  for the treatment of ovarian, breast , and panmaxC) of olaparib in patients reached 14.2 µM at the recommended dosage of 400 mg . HCT116 cells (3.0 × 105 cells/well) were seeded into a 24-well plate for 24 h. After reaching a confluence of 60%, the transfection mixture containing 0.25 µg of DNA (diluted in Opti-MEM medium) and 0.75 μL of JetPrime transfection reagent was incubated for 10 min at room temperature, according to the supplier’s instructions. Meanwhile, culture media was replaced with a fresh supply. The transfection solution was added dropwise into the wells, and transfected cells were incubated for 24 h before being used for AKR1C3 inhibitory studies or drug combinations. The uniformity of transfection and AKR1C3 expression were monitored as previously described [The pCI_AKR1C3 plasmid encoding the AKR1C3 enzyme and pCI Empty Vector (EV) was propagated in the escribed .2). Thereafter, cells were lysed with 200 μL lysing buffer  for 15 min at room temperature. Drug solutions and vehicle controls were mixed with respective lysates, and formed Daun-ol was extracted twice using ethyl acetate. Following evaporation of the organic phase under the vacuum and dissolution of the dry residues in the mobile phase, Daun-ol concentrations were determined by UHPLC.The AKR1C3 inhibitory activity of olaparib was assessed at the cellular level. Transfected HCT116 cells were established according to the procedure described above. Media containing 5 μM Daun, with or without olaparib solution, was added to each well . The drug solutions and vehicle controls were harvested after 2 and 4 h incubation at standard conditions . Cells were treated with drugs or a vehicle for the time intervals specified in their respective subsections. Cellular proliferation was assessed using a detection solution composed of XTT (1 mg/mL) in Opti-MEM medium and phenazine methosulphate (7.5 mg/mL). The HCT116, HepG2, and KG1α cells were incubated with the detection solution for 1 h, 2 h, and 3 h, respectively. Cellular absorbance was measured at 450 nm using a microplate reader .4 cells/well) and HepG2 (1.8 × 104 cells/well) cells were seeded on 96-well plates. After 24 h, transfected HCT116 cells and HepG2 cells were incubated with Daun (range of 0.01–1.00 µM) with or without 5 or 10 µM olaparib. As well as the combination, the toxicity of olaparib alone was also assessed . KG1α (2.5 × 104 cells/well) cells were seeded and immediately treated with Daun (0.010–0.250 µM) with or without 5 or 10 µM olaparib. In addition, olaparib toxicity alone was assessed in a range of 0.05–50.00 µM. For all cell lines, viability was determined after 72 h of incubation at standard conditions using an XTT assay (see the previous subsection). Combination effects were quantified according to a combination index (CoI) from the Chou–Talalay method [PARP1 and PARP2 mRNA expression was monitored in transfected HCT116 sublines using qRT-PCR. Absolute expression of target genes was quantified using TaqMan probes, TaqMan Universal Master Mix II (no UNG), and qRT-PCR standards, according to manufacturer’s instructions. RNA isolation and cDNA preparation were performed as described in the following section.Transfected HCT116 (2.5 × 10y method . Next toAKR1C3 expression. HepG2 (30 × 104) and KG1α (25 × 104) were seeded into 12-well plates . At time 0, cells were treated with 1 or 5 µM olaparib or vehicle control; then, samples were collected at 24 and 48 h intervals. For RNA extraction, cells were lysed with TriReagent, and total RNA was extracted using Zymo Research’s Direct-Zol RNA mini prep kit. The cDNA was generated as described previously [AKR1C3 mRNA in samples was quantified using AKR1C3 standard, which was prepared as described previously [AKR1C3-specific primers (1 µM), and 20 ng cDNA. The qPCR was performed using a QuantStudio 6flex  with the following conditions: initial denaturation at 95 °C for 10 min, then 40 cycles at 95 °C for 15 s and 65 °C for 1 min.First, olaparib cytotoxicity was screened in model cell lines to estimate a suitable concentration for use in induction studies. HepG2 and KG1α cells were seeded at densities identical to those used in the drug combination studies. Cells were treated with olaparib on a six-point concentration range (0.05–20.00 µM) and incubated for 48 h at standard conditions. Cell viability was assessed using an XTT proliferation assay. Subsequently, induction studies based on qRT-PCR analysis were performed to determine the effect of olaparib on eviously . AKR1C3 pG2 30 × 4 and KG1Daun-ol concentrations were determined using a UHPLC Agilent 1260 Series chromatographic system equipped with a dual-fluorescence detector . The enzyme reaction mixture was pre-filtered through a 0.2 μm polytetrafluoroethylene (PTFE) syringe filter, and then injected into a Zorbax Eclipse Plus C18 RR HD column  with a 1290 Infinity inline filter . The mobile phase consisted of a mixture containing formic acid (0.1%), water (74%), and acetonitrile (26%). The flow rate was 0.7 mL/min. Eluents were detected using a fluorescence detector at excitation and emission wavelengths of 480 and 560 nm, respectively.p-values < 0.05 were considered statistically significant. One-way ANOVA followed by Dunnett’s post-hoc test or a two-tailed unpaired t-test were used for p-value calculations, as specified in particular figure legends. Combination indices (CoI) were calculated using CompuSyn 3.0.1 software .Statistical analysis was performed in GraphPad Prism 8.0.4 ; AKR1C3 expression in either systemic or intratumoral models, and thus does not exhibit the potential to establish systemic Daun resistance or counteract the beneficial combination effect. Our observations represent valuable knowledge that could be transformed into more effective therapies in AKR1C3-expressing tumours.In conclusion, we demonstrated that olaparib potently inhibits the AKR1C3 enzyme at clinically relevant concentrations. This interaction was proven to attenuate Daun resistance, and can be assumed to participate on the synergistic outcome of olaparib combined with anthracyclines. Advantageously, olaparib did not affect


Cancer cell dissemination is facilitated by actin-rich plasma membrane protrusions called invadopodia, which focally degrade matrix tissues. Zagryazhskaya-Masson et al. show that invadopodia formation and function depend on the interaction between the scaffolding protein, TKS5, and the CDC42 guanine exchange factor, FGD1. Tumor cells exposed to a physiological matrix of type I collagen fibers form elongated collagenolytic invadopodia, which differ from dotty-like invadopodia forming on the gelatin substratum model. The related scaffold proteins, TKS5 and TKS4, are key components of the mechanism of invadopodia assembly. The molecular events through which TKS proteins direct collagenolytic invadopodia formation are poorly defined. Using coimmunoprecipitation experiments, identification of bound proteins by mass spectrometry, and in vitro pull-down experiments, we found an interaction between TKS5 and FGD1, a guanine nucleotide exchange factor for the Rho-GTPase CDC42, which is known for its role in the assembly of invadopodial actin core structure. A novel cell polarity network is uncovered comprising TKS5, FGD1, and CDC42, directing invadopodia formation and the polarization of MT1-MMP recycling compartments, required for invadopodia activity and invasion in a 3D collagen matrix. Additionally, our data unveil distinct signaling pathways involved in collagenolytic invadopodia formation downstream of TKS4 or TKS5 in breast cancer cells. Migration of cells through tissues is essential during embryonic development, tissue repair, and immune surveillance . Deregul2; Studies using tumor cells plated on a thin layer of gelatin revealed that invadopodia formation is a multistep process initiated by the assembly of F-actin and cortactin-positive invadopodia precursors . PrecursRecent work revealed that invadopodia structure and activity differ depending on the composition and mechanical properties of the matrix environment . In the 3/4C antibody that detects the collagenase-cleaved fragment of collagen I showed a robust collagenolytic activity associated with TKS5GFP-positive structures  analysis was performed using MDA-MB-231 cells that did not express TKS5GFP as a negative control. Several proteins that were specifically identified in the immunoprecipitates from TKS5GFP-expressing cells had been already identified as TKS5 partners, some with known invadopodia localization and/or function, validating our approach  and SH3#5 (W1092A) domains of TKS5 (#5 (W1092A) mutant retained only 10% of the wild-type TKS5 binding capacity (#5 domain (as well as the double W861A/W1092A mutation) severely affected the colocalization of TKS5 with FGD1  compartments, which congregate near the centrosome located ahead of the nucleus during the invasion of MDA-MB-231 cells in a dense 3D collagen fibrillary matrix . In addimovement . Next, wmovement . We confagen gel . In addiagen gel . In addiagen gel , in agreagen gel . Thus, wagen gel . Noticeaagen gel . Finallyagen gel  and globagen gel . AltogetGFP, whereas TKS4 was not , interacts with the amino-terminal proline-rich domain of FGD1 (aa 1–335). Specifically, we found that mutation of a critical W residue in SH3#5 -trisphosphate (CAS (CAS accumulated together with cortactin in invadopodia in association with the collagen fibers (2 into PI(3)P, was enriched in TKS5-positive invadopodia, probably by binding to its PIP2 substrate  and insert more MT1-MMP in the invadopodial plasma membrane in a feed-forward invadopodia maturation loop P2 production at early contact sites between surface-exposed MT1-MMP and type I collagen fibrils, preceding the expansion and stabilization phase of invadopodia structures  such that the first in a conserved pair of W residues was converted to A to generate TKS5GFPW861A, TKS5GFPW1092A, and TKS5GFPW861A/W1092A (the position of the mutations is based on human TKS5 containing the PX domain). TKS5 SH3#4 forward primer: 5′-GAAGCAGGAGAGAGCGGGGCGTGGTATGTGAGGTTT-3′, and reverse primer: 5′-AAACCTCACATACCACGCCCCGCTCTCCTGCTTC-3′; TKS5 SH3#5 forward primer: 5′-GAGGAACCCTAATGGCGCGTGGTACTGCCAGATC-3′, and reverse primer: 5′-GGCAGTACCACGCGCCATTAGGGTTCCTCTCCAG-3′. The TKS4GFP construct was obtained by cloning the insert in the SacI and BamH1 sites of the pEGFP-N1 vector. The sequences corresponding to three regions of FGD1 encompassing the entire FGD1 protein were prepared from the pEGFP-C1-FGD1 construct and cloned into pET-MCn-His-GST vector to make GST–FGD1 fusion proteins. The N-term domain  was cloned into NdeI and XhoI sites, the middle  was cloned into NdeI and BamHI sites, and the carboxy-terminal domain  was cloned into NdeI and BamHI sites of the receiving vector. The GFP-tagged FGD1 construct deleted of the PRD domain  was obtained by site-directed mutagenesis of the pEGFP-C1-FGD1 plasmid by PCR using the following primers: forward, 5′-CTCAAGCTTCGAATGACAGTGACCTGG-3′, and reverse, 5′-GGTCACTGTCATTCGAAGCTTGAGC-3′. PCR products were digested by Dpn1 followed by transformation into XL1-Blue cells. Plasmids isolated from the resulting colonies were screened for the desired modification by digestion with AflII and XhoI. All constructs were verified by sequencing before use.Plasmids used for this study and sources are listed in 2. Hs578T cells  were grown at 37°C under 5% CO2 in DMEM GlutaMAX  supplemented with 10% FCS , 5 mM pyruvate , and 1% penicillin-streptomycin . HT-1080 fibrosarcoma cells  were grown in DMEM GlutaMAX supplemented with 10% FCS. Cells were routinely tested for mycoplasma contamination. MDA-MB-231 cells stably expressing TKS5GFP were generated by lentiviral transduction (5 cells were plated in a 24-well plate. The transfection mixture containing Lipofectamine 3000 together with P3000 reagent (Thermo Fisher Scientific) and 0.5–1 µg of a plasmid was added immediately after cell plating, and the transfection mixture was replaced with fresh growth medium 15 h later. For RNA interference, MDA-MB-231 cells were treated with the indicated siRNAs (50 or 100 nM) using Lullaby (OZ Biosciences) and analyzed after 72 h of treatment. Hs578T cells were treated with indicated siRNAs using Lipofectamine 3000 for 72 h before analysis according to the manufacturer’s instructions, with minor modifications. Briefly, the cells were trypsinized, 40,000 cells were plated per well of 24-well plate, and the Lipofectamine 3000 mixture containing 100 nM of siRNA was added immediately. The transfection mixture was replaced with fresh growth medium 15 h later. siRNAs used for this study are listed in MDA-MB-231 cells  were grown in L15 medium supplemented with 15% FCS and 2 mM glutamine at 37°C in 1% COsduction . For tra7 cells per dish), and 20 h later, cells were lysed in NP-40 buffer as described for the immunoprecipitation procedure. GST or GST-tagged FGD1 protein fragments (2 µM) were immobilized onto Glutathione Sepharose 4B beads  in binding buffer  for 1 h at 4°C under constant agitation. The beads were washed three times with the washing buffer and incubated with the cell supernatants for 2 h at 4°C under constant agitation. At the end of the procedure, the beads were washed twice with the washing buffer and then twice with the binding buffer. The bound proteins were eluted in Laemmli buffer by heating to 95°C for 10 min and analyzed by SDS-PAGE.Cells were transfected with plasmids encoding the indicated constructs using Lipofectamine 3000  or not (MDA-MB-231) were plated on a layer of rat collagen type I for 90 min , and cells were collected, lysed in NP-40 buffer  for 30 min at 4°C under constant agitation. The lysate was centrifuged at 15,000 g for 10 min at 4°C, and the supernatant was incubated with 25 µl of equilibrated control magnetic agarose beads (ChromoTek) for 1 h at 4°C under constant agitation. The precleared lysate was then incubated with 25 µl of equilibrated magnetic agarose beads coupled to anti-GFP nanobodies  for another 1 h at 4°C. At the end of the procedure, GFP-Trap beads were washed three times with washing buffer , followed by four washes with dilution buffer  and collected in 200 µl of dilution buffer for overnight storage at 4°C before MS analysis. In immunoprecipitation experiments followed by immunoblotting analysis, after the final wash, the beads were immediately heated at 95°C for 10 min under constant agitation in Laemmli buffer and stored at −20°C before immunoblotting analysis.Cells stably expressing TKS54HCO3, and on-beads digestion was performed with 0.2 µg trypsin/LysC (Promega) for 60 min in 100 µl of 25 mM NH4HCO3. Sample were then loaded onto a homemade C18 StageTips for desalting. Peptides were eluted using 40/60 MeCN/H2O + 0.1% formic acid and vacuum concentrated to dryness.Proteins on magnetic beads were washed twice with 100 µl of 25 mM NH2O in 0.1% formic acid) at a flow rate of 4.0 µl/min over 4 min. Separation was performed on a 50 cm × 75 µm C18 column  regulated to a temperature of 55°C with a linear gradient of 5–25% buffer B (100% MeCN in 0.1% formic acid) at a flow rate of 300 nl/min over 100 min. Full-scan MS was acquired in the Orbitrap analyzer with a resolution set to 120,000, and ions from each full scan were fragmented by high-energy collisional dissociation and analyzed in the linear ion trap.Online chromatography was performed with an RSLCnano system  coupled online to an Orbitrap Fusion Tribrid mass spectrometer (Thermo Fisher Scientific). Peptides were trapped on a C18 column  with buffer A (2/98 MeCN/HHF through Proteome Discoverer (v2.1). Enzyme specificity was set to trypsin, and a maximum of two missed cleavage sites were allowed. Oxidized methionine, amino-terminal acetylation, and carbamidomethyl cysteine were set as variable modifications. Maximum allowed mass deviation was set to 10 ppm for monoisotopic precursor ions and 0.6 D for MS/MS peaks.For identification, the data were searched against the human SwisProt database (February 2017) using SequestThe resulting files were further processed using myProMS v3.6 . False dCells were lysed in SDS sample buffer, separated by SDS-PAGE, and detected by immunoblotting analysis with the indicated antibodies. Antibodies were visualized using the ECL detection system .2 before fixation. Cells were preextracted with 0.1% Triton X-100 in 4% PFA in PBS during 90 s, fixed in 4% PFA in PBS for 20 min, and stained for immunofluorescence microscopy with indicated antibodies.Coverslips were layered with 200 µl ice-cold 2.0 mg/ml acidic extracted collagen I solution (Corning) in 1× MEM mixed with 4% Alexa Fluor 647–conjugated type I collagen. The collagen solution was adjusted to pH 7.5 using 0.34 N NaOH, and Hepes was added to 25 µM final concentration. After 3 min of polymerization at 37°C, the collagen layer was washed gently in PBS, and cells in suspension were added for 60–90 min at 37°C in 1% CO4 cells were plated on type I collagen–coated coverslips, fixed after 60 min, and stained with TKS5 or FGD1 pAbs and counterstained with cortactin mAb. Images were acquired with a wide-field microscope  using a 100× Plan Apo VC 1.4 oil objective and a highly sensitive cooled interlined charge-coupled device camera . A z-dimension series of images was taken every 0.2 µm by means of a piezoelectric motor (Physik Instrumente). For quantification of TKS5 or FGD1 associated with invadopodia, three consecutive z-planes corresponding to the plasma membrane in contact with collagen fibers were projected using maximal-intensity projection in Fiji, and TKS5 or FGD1 signal was determined using the thresholding command excluding regions <8 px to avoid noninvadopodial structures. Surface covered by TKS5 or FGD1 signal was normalized to the total cell surface, and values were normalized to those of control cells.5 × 10For quantification of the colocalization between the invadopodial markers the along collagen fibers, linescans were performed using Fiji software. Briefly, a segmented line (6-pixel width) was drawn along collagen fibers that were associated with the invadopodial markers . The fluorescence intensity signal of each pixel was measured, normalized to the maximal-intensity signal set to 100, and plotted on an x-y graph where the x axis represents the length of the invadopodia (in pixels) and the y axis represents the normalized fluorescence intensity of the markers. Correlation between the two markers was observed by plotting the normalized values of each invadopodial marker on a graph in which the x axis represents the normalized fluorescence intensity of one marker and the y axis represents the normalized fluorescence intensity of the other marker. Linear regression line was calculated using GraphPad Prism software.Colocalization analyses were performed on Fiji software based on two tools . First, 5 cells/ml) in 200 µl of ice-cold 2.0 mg/ml collagen I solution prepared as previously described. 40 µl of the cell suspension in collagen was added on a glass coverslip, and collagen polymerization was induced for 30 min by incubation at 37°C. L-15 complete medium was then added, and cells embedded in collagen were incubated for 16 h at 37°C in 1% CO2. After fixation for 30 min at 37°C in 4% PFA in PBS, samples were incubated with anti-Col1-3/4C antibodies for 2 h at 4°C. After extensive washes, samples were counterstained with Cy3-conjugated anti-rabbit IgG antibodies and Phalloidin-Alexa Fluor 488 to visualize cell shape and mounted in DAPI. Image acquisition was performed with an A1R Nikon confocal microscope with a 40× NA 1.3 oil objective using a high-sensitivity 455-nm GaASP PMT detector and a 595 ± 50-nm bandpass filter. Quantification of degradation spots was performed as previously described (Cells treated with indicated siRNAs were trypsinized and resuspended (2.5 × 10escribed . Briefly5 cells/ml) in complete medium was added and incubated for 30 min at 37°C. Medium was gently removed, and two drops of a mix of Alexa Fluor 647–labeled type I collagen/unlabeled type I collagen at 2.0 mg/ml final concentration were added on top of the cells (top layer). After polymerization at 37°C for 90 min as described above, 2 ml of medium containing 20 ng/ml HGF was added to the culture. Z-stacks of images were acquired every 10 min during 16 h by confocal spinning disk microscopy (Roper Scientific) using a CSU22 Yokogawa head mounted on the lateral port of an inverted TE-2000U Nikon microscope equipped with a 40× 1.4-NA Plan-Apo objective lens and a dual-output laser launch, which included 491- and 561-nm, 50-mW DPSS lasers (Roper Scientific). Images were collected with a CoolSNAP HQ2 charge-coupled device camera (Roper Scientific). The system was steered by Metamorph 7 software. Kymographs were obtained with Fiji software along a line spanning the invadopodia diameter.Glass-bottom dishes (MatTek Corp.) were layered with 10 µl of a solution of 5 mg/ml unlabeled type I collagen mixed with 1/25 volume of Alexa Fluor 647–labeled collagen. Polymerization was induced at 37°C for 3 min as described above, and the bottom collagen layer was washed gently in PBS. 1 ml of cell suspension (10A homemade Matlab program (available on demand) was developed to track nuclei based on nuclear staining and create a velocity-dependent coordinate system to analyze MT1-MMP endosomes relative to the direction of displacement of the nucleus . Nuclei 200 µl of 2.0 mg/ml collagen I was allowed to polymerize in Transwell inserts (Corning) for 2 h at 37°C as above. Cells were seeded on top of the collagen gel in complete medium, and 20 ng/ml HGF was added to the medium in the bottom chamber of the Transwell as chemoattractant. After 48 h of seeding, cells were fixed and stained with DAPI and visualized by confocal microscopy with serial optical sections captured at 10-µm intervals with a 10× objective on a Zeiss LSM510 confocal microscope. Invasion was measured by dividing the sum of DAPI signal intensity of all slides beyond 30 µm (invading cells) by the sum of the intensity of all slides .U tests were applied as indicated in the figure legends.All results were presented as mean ± SEM of three independent experiments. GraphPad Prism was used for statistical analysis. Statistical significance was defined as *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; and ns, not significant. Data were tested for normal distribution using the D’Agostino–Pearson normality test, and nonparametric tests were applied otherwise. One-way ANOVA, Kruskal–Wallis, and Mann–Whitney GFP and FGD1, while coimmunoprecipitation is detected between TKS5GFP and FGD1, as well as a comparison of the collagenolysis activity of MDA-MB-231 and Hs578T cells in a 3D collagen gel. GFP and TKS5mCherry plated on a fibrillary type collagen layer, documenting the dynamics of the double-positive invadopodia structures. GFP identified by coimmunoprecipitation and MS analysis. The interactions of TKS5 with disintegrin and metalloproteinase domain–containing protein 15, monocarboxylate transporter 4, 14–3-3 family proteins, and FGD1 were previously reported (reported . Table SReview HistoryClick here for additional data file.Table S1GFP-expressing MDA-MB-231 cells versus wild-type MDA-MB-231 cells.lists proteins corresponding to the peptides quantified by LC-MSMS in GFP-Trap immunoprecipitates from TKS5Click here for additional data file.Table S2lists antibodies used in this study.Click here for additional data file.Table S3lists siRNAs and plasmids used in this study.Click here for additional data file.


LIN28B distal enhancer DNA sequence, which contains three potential OCT4 target sequences. By a combination of chemical mapping and cryo-electron microscopy single-particle analysis, we mapped the positions of the three target sequences within the nucleosome. A mutational analysis revealed that OCT4 preferentially binds its target DNA sequence located near the entry/exit site of the nucleosome. Crosslinking mass spectrometry consistently showed that OCT4 binds the nucleosome in the proximity of the histone H3 N-terminal region, which is close to the entry/exit site of the nucleosome. We also found that the linker histone H1 competes with OCT4 for the nucleosome binding. These findings provide important information for understanding the molecular mechanism by which OCT4 binds its target DNA in chromatin.Transcription factor binding to genomic DNA is generally prevented by nucleosome formation, in which the DNA is tightly wrapped around the histone octamer. In contrast, pioneer transcription factors efficiently bind their target DNA sequences within the nucleosome. OCT4 has been identified as a pioneer transcription factor required for stem cell pluripotency. To study the nucleosome binding by OCT4, we prepared human OCT4 as a recombinant protein, and biochemically analyzed its interactions with the nucleosome containing a natural OCT4 target, the  In eukaryotes, genomic DNA forms chromatin with the nucleosome as the basic repeating unit, in which a segment containing about 150 base-pairs of DNA is tightly wrapped around a histone octamer3. The nucleosome formation generally restricts the binding of TFs to their target DNA sequences in the genome. However, a group of TFs, called pioneer TFs, are able to bind to their target DNA sequences within the nucleosome by recruiting downstream transcription factors and chromatin remodelers, and consequently these TFs change the chromatin conformation to regulate the transcription of certain sets of genes6. The pioneer TF-mediated transcription then regulates cellular differentiation5.Transcription factors (TFs) induce the initiation of gene transcription by binding to their target DNA sequences in the genome8. At the initial stage of the reprogramming process, OCT4, SOX2, and KLF4 may function as pioneer factors and bind to the “closed chromatin” state, inducing the conversion to the “open chromatin” state9. OCT4, SOX2, and KLF4 efficiently bind to reconstituted nucleosomes in vitro, and preferentially accumulate at genomic DNA regions occupied by nucleosomes in vivo10.Among the pioneer TF family proteins, the overexpression of OCT4, together with SOX2, KLF4, and c-MYC, reportedly promotes the reprogramming of cells to pluripotencyS and POUHD, which are located in the middle of the peptide, bind a specific octamer DNA sequence, ATGC(A/T)AAT, and closely related sequences in chromatin13. A previous crystallographic study revealed that a domain containing the tandem POUS and POUHD of OCT4 binds its target DNA sequence at the major groove15. In the POUS·POUHD-DNA complex, the POUS and POUHD motifs sandwich the DNA, and sterically clash with histone binding to the nucleosomal DNA10. Therefore, OCT4 may partially peel the DNA from the histone surface upon nucleosome binding. However, the detailed mechanism by which OCT4 specifically recognizes its target DNA sequence in the nucleosome has not been elucidated.Human OCT4 is composed of 360 amino acid residues, and two DNA-binding motifs, POULIN28B distal enhancer DNA sequence, which contains three potential OCT4 target DNA sequences. The LIN28B gene encodes a paralog of the Lin28 protein, Lin28B, which is crucial for reprogramming and pluripotency18. We found that OCT4 specifically recognizes its target DNA sequence, which is located at the entry/exit site of the nucleosome. A crosslinking mass spectrometry analysis revealed that the OCT4 bound to the nucleosome is located in the proximity of the N-terminal region of histone H3, which is also near the entry/exit site of the nucleosome. Finally, we found that the linker histone H1 competes with OCT4 for nucleosome binding. These new findings provide novel insights toward understanding the molecular mechanism by which OCT4 binds chromatin and regulates the pluripotency of cells.In the present study, we reconstituted the nucleosome with the native LIN28B distal enhancer region (LIN28B nucleosome). Three potential OCT4 target sequences  exist in the 162 base-pair DNA fragment . The LIN28B nucleosome was mainly positioned at the center of the 162 base-pair DNA fragment with linker DNAs on both sides , although the other positions were also observed as smeared bands  intrinsically possesses a nucleosome positioning property.We reconstituted the LIN28B nucleosome by the salt dialysis method in vitro, and tested its nucleosome positioning by the chemical probing assay. To do so, the LIN28B nucleosome was reconstituted with the human canonical histones H2A, H2B, H3.1, and the histone H4 S47C mutant, in which the Ser47 residue was replaced by Cys Fig. . In the We then determined the structure of the LIN28B nucleosome by cryo-electron microscopy. The OCT4-LIN28B nucleosome complex was prepared by sucrose gradient, and cryo-EM images were collected Fig. . In the 6-tagged human OCT4 (5CS) protein  are replaced with Ser to stabilize the protein21. Consistent with the previous result10, OCT4 efficiently bound to the LIN28B nucleosome, and appeared as a discrete band corresponding to the OCT4-nucleosome complex , and reconstituted the mutant LIN28B nucleosomes  and ten (+ 10) base pairs toward the nucleosomal dyad Fig. . Since sS·POUHD domain of OCT4 was predominantly crosslinked with the N-terminal region of histone H3  analysis, to map the OCT4 binding site relative to the core histones in the nucleosome. The lysine residues located close to the OCT4-nucleosome complex were crosslinked with disuccinimidyl suberate (DSS)-H12/D12. The crosslinked lysine residues between OCT4 and histones were then detected by mass spectrometry Fig. . Intrigu26, and alter the relaxed chromatin conformation to the condensed form27. In pluripotent cells, the chromatin conformation is largely relaxed, allowing the nucleosome binding of the pioneer transcription factors, including OCT429. Interestingly, citrullination of the H1 DNA-binding site reportedly induces H1 displacement and chromatin decondensation in pluripotent cells30. The transition of linker histone types also occurs during early embryogenesis31. This may play an important role in the global conformational change of chromatin to promote the transcriptionally active somatic state during developmental processes. In these processes, the OCT4 bound to the nucleosome may be replaced by linker histones, suppressing the cells back to the pluripotent state. Therefore, we tested whether the OCT4-nucleosome binding could be replaced by the H1 binding, by performing a competitive nucleosome binding assay  chromatin, which is generally inaccessible to DNA binding proteins. Therefore, pioneer TFs somehow bind their target DNA in chromatin to induce the reprogramming. In the present study, we focused on the pioneer TF, OCT4, and studied its specific binding to the LIN28B nucleosome containing its natural binding DNA sequence, the LIN28B distal enhancer DNA sequence contains target DNA sequences for pioneer TFs, including OCT4, SOX2, and KLF4, which are known to promote induced-pluripotent stem cells (iPSC)10. We performed the OCT4 binding assay with the reconstituted LIN28B nucleosomes containing the OCT4 targeting sequence in various positions. Our chemical probing and cryo-EM analyses revealed that the LIN28B distal enhancer DNA fragment forms a positioned nucleosome, and is properly wrapped around the histone octamer  and the 193 base-pair 601 DNA fragment with the OCT4 target sequence  were each cloned into the pGEM-T-Easy vector (Promega), and used as the templates for PCR amplification. Mutations were introduced by the QuikChange system (Stratagene). The DNA fragments were amplified by PCR and purified by non-denaturing polyacrylamide gel electrophoresis, using a Prep Cell apparatus (Bio-Rad). The eluted DNA fragments were collected in 10 mM Tris–HCl (pH 8.0) buffer containing 0.1 mM EDTA, and were concentrated with a Millipore centrifugal filter.The 162 base-pair 6-tagged OCT4 (5CS) was produced in the E. coli strain BL21-CodonPlus(DE3) RIL, by incubating the culture at 18 °C for 20–24 h after the addition of IPTG to a final concentration of 0.5 mM. The cells were harvested and resuspended in buffer A . The cells were sonicated on ice, and the lysate was centrifuged at 39,000g for 20 min at 4 °C. The pellets were resuspended in buffer B , and incubated overnight at 4 °C. The supernatant containing His6-OCT4 (5CS) was mixed with Ni–NTA agarose resin (Qiagen), and the sample was rotated for 1 h at 4 °C. Afterwards, the resin was washed sequentially with buffer C  and buffer C containing 25 mM imidazole, and the OCT4 protein was eluted with buffer C containing 150 mM imidazole. The eluted sample was concentrated with a Millipore centrifugal filter, and the OCT4 protein was refolded by dripping the sample into × 100 volume of buffer D . The OCT4 protein was finally purified by Superdex200  gel filtration column chromatography in buffer E . The purified OCT4 protein was concentrated using a Millipore centrifugal filter, and flash frozen in liquid nitrogen.Human OCT4 (5CS) was expressed from the pET15b vector (Novagen), in which the thrombin protease recognition site was replaced with the PreScission site. The His43. Using the purified, lyophilized histones, the H2A-H2B and H3-H4 complexes were reconstituted and isolated43. The complexes were flash frozen in liquid nitrogen, and stored at − 80 °C.The human histones H2A, H2B, H3.1, and H4 were purified as recombinant proteins, as described previously43. Briefly, a DNA fragment was mixed with the H2A-H2B and H3-H4 complexes in high-salt buffer, and the nucleosomes were reconstituted by the salt dialysis method43. To prepare the nucleosome for the chemical probing assay, H4 S47C, in which the Ser47 residue of H4 was replaced by Cys, was used instead of wild-type H4. The resulting nucleosomes were further purified by non-denaturing polyacrylamide gel electrophoresis, using a Prep Cell apparatus (Bio-Rad). The nucleosomes were collected in buffer F [20 mM Tris–HCl (pH 7.5) and 1 mM DTT], and were concentrated using a Millipore centrifugal filter. The nucleosome samples were stored at 4 °C.The nucleosomes were prepared as described previously2.The OCT4-LIN28B nucleosome complex for the cryo-EM analysis was purified by sucrose gradient ultracentrifugation. A gradient was formed with buffer G  and buffer G containing 15% sucrose, using a gradient maker. For complex formation, the LIN28B nucleosome (0.96 µM) was mixed with OCT4 (nucleosome:OCT4 = 1:6 molar ratio) in buffer H , and was incubated for 1 h on ice. The sample was applied on the top of a gradient, and was centrifuged at 27,000 rpm at 4 °C for 16 h, using a Beckman Sw41Ti rotor. The fractions were analyzed by non-denaturing polyacrylamide gel electrophoresis, and the peak fractions were dialyzed against buffer F. The sample was then concentrated using a Millipore centrifugal filter. Tween-20 was added to a final concentration of 0.00074%, and a 2 µl portion of the sample (0.6 mg/ml) was applied to a glow-discharged Quantifoil holey carbon grid (R1.2/1.3 200-mesh Cu). The grids were blotted for 6 s under 100% relative humidity at 16 °C, and were immediately plunged into liquid ethane, using a Vitrobot Mark IV (Thermo Fisher). Cryo-EM images were collected by the EPU auto acquisition software on a Talos Arctica cryo-electron microscope (Thermo Fisher), operated at 200 kV at a nominal magnification of × 100,000, which renders a pixel size of 1.32 Å at the object scale. Images were recorded under low-dose conditions with 10-s exposure times, using a K2 Summit direct electron detector and a GIF Quantum energy filter (slit width 20 eV) (Gatan) in the counting mode, retaining a total of 40 frames with a total dose of ~ 50 electrons per Åhttps://emcore.ucsf.edu/ucsf-motioncor2)44, with dose weighting. The contrast transfer function (CTF) was estimated by CTFFIND4 (https://grigoriefflab.janelia.org/ctf)45 from the digital micrographs, with dose weighting. In total, 1,274 images were selected based on the CTF fit correlation to approximately 5 Å resolution. RELION 3.0 (https://www2.mrc-lmb.cam.ac.uk/relion/index.php/Main_Page)46 was used for all subsequent image processing operations. Subsequently, 325,272 particles of the LIN28B nucleosome were picked automatically, with a box-size of 150 × 150 pixels. Two-dimensional classification to remove bad particles resulted in the selection of 264,297 particles. The crystal structure of a canonical nucleosome (PDB: 3LZ0) in the low-pass filtered to 60 Å was used as the initial three-dimensional reference. The best classes containing 150,721 particles, in which the nucleosomal DNA was fully wrapped around the histone octamer, were selected from the three-dimensional classification. The three-dimensional refinement of the LIN28B nucleosome was performed, followed by particle polishing and two rounds of CTF refinement. The final three-dimensional map of the LIN28B nucleosome was sharpened with an exponential B-factor (− 50.7 Å2). The gold standard Fourier Shell Correlation (FSC) at the FSC = 0.143 criterion47 was used for the resolution estimation of the refined map. The local resolution map of the LIN28B nucleosome was calculated with ResMap (https://resmap.sourceforge.net)48. The final three-dimensional map was normalized with MAPMAN (https://xray.bmc.uu.se/usf/mapman_man.html)49, and the iso-electron potential surface of the LIN28B nucleosome was visualized with UCSF Chimera (https://www.cgl.ucsf.edu/chimera/)50.In total, 1,877 movies of the LIN28B nucleosome were aligned and integrated using MOTIONCOR2 (43. The LIN28B nucleosome containing H4 S47C (37.4 pmol) was labeled with N- iodoacetamide at a molar ratio of 1:50 (nucleosome:iodoacetamide) in buffer I [10 mM Tris–HCl (pH 7.5) and 50 mM NaCl] overnight at 4 °C. To remove the excess label, the sample solution was exchanged with buffer J [50 mM Tris–HCl (pH 7.5) and 2.5 mM NaCl], using a Millipore centrifugal filter. CuCl2 was added to a final concentration of 0.15 mM, and the sample was incubated for 2 min at room temperature. The sample solution was then exchanged with buffer J, using a Millipore centrifugal filter. To initiate the reaction, MPA (3-mercaptopropionic acid) and H2O2 were added to a final concentration of 6 mM each, and the sample was incubated for 5 min at room temperature. To stop the reaction, neocuproine was added to a final concentration of 2.8 mM. The DNA fragments were then extracted with phenol–chloroform, and precipitated with ethanol. The purified DNA fragments were resuspended in Hi-Di Formamide, and incubated at 95 °C for 5 min. The DNA samples were analyzed by electrophoresis on a denaturing 10% polyacrylamide gel containing 7 M urea. The DNA bands were visualized by ethidium bromide staining.The in vitro chemical probing assay was performed according to the previously reported method6-OCT4 (5CS). The samples were incubated for 1 h on ice, in a solution containing 2 mM Tris–HCl (pH 7.5), 13 mM HEPES–NaOH (pH 7.5), 50 mM NaCl, 0.1 mM DTT, 2.2 mM β-mercaptoethanol, and 0.03% NP-40. After the incubation, the reaction products were analyzed by non-denaturing polyacrylamide gel electrophoresis with ethidium bromide staining.Each reaction was performed in a total volume of 10 µl. The LIN28B nucleosome (0.1 µM) was mixed with 0 µM, 0.1 µM, or 0.2 µM of HisEach reaction was performed in a total volume of 10 µl. For the OCT4 titration experiments, the 193 base-pair 601 nucleosome containing the OCT4 target sequence  was preincubated with or without the linker histone H1.2  for 20 min on ice. OCT4  was then added and the mixture was further incubated for 1 h on ice. For the H1 titration experiments, the 193 base-pair 601 nucleosome containing the OCT4 target sequence  was preincubated with or without OCT4 . H1.2  was then added and the mixture was further incubated for 1 h on ice. The final reaction solution contained 30 mM Tris–HCl (pH 7.5), 1.5 mM HEPES–NaOH (pH 7.5), 65 mM NaCl, 0.1 mM DTT, 2.9 mM β-mercaptoethanol, 0.001% PGA, and 4% glycerol. The reaction mixtures were analyzed by non-denaturing polyacrylamide gel electrophoresis with ethidium bromide staining.52. The LIN28B nucleosome (4.3 μM) was mixed with OCT4 in a nucleosome:OCT4 = 1:4 molar ratio, and was incubated on ice for 1 h. The sample was then crosslinked with 9.6 mM DSS-H12/D12 (Creative Molecules) at 30 °C for 30 min. The reaction was stopped by adding 48 mM ammonium bicarbonate, and then incubated at 30 °C for 15 min. The sample was reduced, alkylated, and then digested by sequencing-grade endopeptidase Trypsin/Lys-C Mix (Promega), at an enzyme–substrate ratio of 1:50 wt/wt. The digested sample was applied to a Superdex 30 Increase 3.2/300  column, using buffer containing 25% acetonitrile and 0.1% TFA. The eluted fractions (150 μl) were collected, dried, and re-dissolved in 0.1% TFA. The samples were analyzed by liquid chromatography tandem mass spectrometry (LC/MS–MS), using an LTQ-Orbitrap Velos mass spectrometer (Thermo Fisher Scientific) equipped with a Zaplous Advance nano UHPLC HTS-PAL xt System (AMR). The crosslinked peptides were identified using the xQuest/xProphet software (https://proteomics.ethz.ch/)51, and the crosslinks were visualized using the webserver xVis (https://xvis.genzentrum.lmu.de/login.php)53. The mass spectrometry raw data used in this study have been deposited to the proteomeXchange Consortium via the JPOST repository 54.Crosslinking mass spectrometry was performed as described previouslySupplementary Information.


Moreover, the incorporation of MoC into the composites inhibits the overgrowth of MoSe2 nanosheets on the 3D carbon framework, producing much smaller MoSe2 nanodots. The obtained MoSe2 nanodots with fewer layers, rich edge sites, and heteroatom doping ensure the good kinetics to promote pseudo-capacitance contributions. Employing as anode material for lithium-ion batteries, it shows ultralong cycle life (with 90% capacity retention after 5000 cycles at 2 A g−1) and excellent rate capability. Moreover, the constructed LiFePO4//MoSe2/MoC/N–C full cell exhibits over 86% capacity retention at 2 A g−1 after 300 cycles. The results demonstrate the effectiveness of the interface engineering by incorporation of MoC as interface bridging intermediate to boost the lithium storage capability, which can be extended as a potential general strategy for the interface engineering of composite materials.Interface engineering has been widely explored to improve the electrochemical performances of composite electrodes, which governs the interface charge transfer, electron transportation, and structural stability. Herein, MoC is incorporated into MoSeThe online version of this article (10.1007/s40820-020-00511-4) contains supplementary material, which is available to authorized users. The XRD patterns suggest the growth of MoSe2 may be changed. No detection of XRD peaks for Mo metal indicates the easier formation of MoC and its stable state at 800 °C.More specific characterizations of material are comprehensively presented within Figs. irection . Unlike −1  analysis as shown in Fig. S1, and the residual product (MoO3) is verified by the XRD pattern in the inset. Furthermore, the XPS spectrum shows the existence of Mo, Se, C, and N elements . The Mo 3d XPS spectrum  in the PVP. Previous experiments and theoretical calculations have shown that Mo–C or Mo–O–C bonds in MoSe2/C nanocomposite could facilitate the ion and electron transportation in the composite [3/2 and 3d5/2 peaks of Se2− in the MoSe2/MoC/N–C nanocomposite  and could be deconvoluted as graphitic N at 400.9 eV, pyrrolic-N at 399.5 eV, and pyridinic-N at 398.3 eV [As for the Raman characterization, all the samples present two bands at around 240 and 280 cmed MoSe2 , 33, 34.ectively , 35, 36.omposite . For the398.3 eV .2 agglomerates into irregular bulks about 20 μm in width. However, as expected, both MoSe2/C and MoSe2/MoC/N–C possessed a highly interconnected three-dimensional porous network with microstructure  of 34.9 m2 g−1 and MoSe2@N–C possesses specific surface area of 21.5 m2 g−1, both of which are much larger than 7.9 m2 g−1 of bare MoSe2  are smaller and more dispersed than those in MoSe2/N–C (600 °C). In comparison, for MoSe2/MoC/N–C, the interconnected 3D porous architecture , much less than that of MoSe2/C. Furthermore, the layer distance of MoSe2/MoC/N–C (0.676 nm) is also larger than that of MoSe2/C (0.647 nm), which facilitate fast Li-ion transportations. Notably, the interlayer distances of 0.25 nm marked in Fig. 2 crystals. And the corresponding fast Fourier transform (FFT) result , the in situ formed MoC is bonded to carbon framework, which can be confirmed by Mo-C and Mo–O-C bonding from XPS  image Fig. g, k revers about 0 layers,2/C and MoSe2/MoC/N–C composites , resulting in few-layered and small-sized MoSe2 nanodots. Furthermore, the in situ formed MoC can pin heterostructure, preventing crystals form and agglomerating. The dark-field scanning TEM and the corresponding elemental mapping images of MoSe2/MoC/N–C reveal that Mo, Se, O, N, and C are evenly distributed on the ultrathin carbon wall  curves obtained during the initial 3 cycles (at a scanning rate of 0.1 mV s−1) exhibit multiple redox reactions for MoSe2/MoC/N–C electrode  of 80.1%, which is higher than most reported MoSe2 electrodes [The MoSe  and bare MoSe2 (440 mAh g−1). This result demonstrates that constructing interconnected 3D porous carbon-modified nanocomposite can indeed improve the capability of storage Li ion. By the way, in the early stages of the cycling  shows that MoC/C possesses the similar morphology to MoSe2/MoC/C composites  according to Table S1, indicating that MoC possess relatively low contents in the electrodes. Therefore, the direct capacity contribution from MoC to the whole electrode is relatively limited. However, MoC plays an important role in the interfacial structure regulation, including more stable chemical binding and reduced MoSe2 particle size, which is more correlated with the rate performance and structural stability. MoSe2/MoC/N–C shows a higher capacity than MoSe2/C mainly because its smaller MoSe2 nanodots can provide more lithium storage sites. The MoC connected MoSe2 and N–C network through C/MoC/MoSe2 multiphase boundaries, enabling fast electron/ion transport and structural stability through interface, resulting in good rate performance and structural stability.To confirm the role of MoC in lithium storage, three-dimensional porous MoC/C was prepared through a similar routine of preparing MoSe2/MoC/N–C electrode in half cell, we assembled a lithium-ion full by pairing with the commercial LiFePO4 cathode  and EIS spectroscopy. The electrochemical kinetics in the as-prepared electrodes could be considered as capacitive-dominated and diffusion-controlled from the CV curves  reveals a faster ionic transportation which resulted in a better rate performance  results  demonstrated that charge transfer resistances (Rct) of MoSe2/MoC/N–C (43 Ω) are smaller than those of MoSe2/C (72 Ω) and bare MoSe2 (557 Ω), suggesting that MoSe2/MoC/N–C shows the fastest interface kinetics. All above analysis suggested that the optimized interconnected 3D porous N–C framework and stronger electronic coupling at interface through MoSe2/MoC/N–C connection facilitate reaction kinetics.The diffusion-controlled reaction kinetics of samples were further analyzed in depth by GITT . As shown in Fig. 2/MoC/N–C electrode after certain cycles at 2 A g−1. Compared to the ex situ TEM images of as-synthesized MoSe2/C , the MoSe2 flakes in the MoSe2/C electrode  and smaller size (about 5 nm). The interlayer distance also expanded to 0.751 nm after cycling. In addition, large areas of graphitized carbon were observed with an enlarged spacing (about 0.411 nm), slightly larger than the initial value of 0.370 nm for the pristine carbon. The expanded interlayer distance of MoSe2 and carbon could be caused by the repeated insertion/extraction of Li ions [2 nanocrystal can further provide Li storage sites, benefiting the capacity of electrode. And the increased graphitization carbon layer enhanced the electronic conductivity. These changes of electrodes material during cycling result in climbing capacities. To further examine the reinforced structure stability of MoSe2/MoC/N–C, the TEM images of MoSe2/MoC/N–C electrode after 500 cycles are compared in Fig. S13B5–B7. Figure S13B5 confirms that the MoSe2/MoC/N–C electrode material retains integrated. As shown in Fig. S13B6 and B7, small shadow dots were still distributed in the carbon framework in MoSe2/MoC/N–C electrode, implying the products were also evenly constrained in carbon framework. These results prove that MoSe2/MoC/N–C connection can constrain the in situ generated MoSe2 and facilitate the highly reversible conversion during cycling.Ex situ TEM images were employed to investigate the structural integrity of MoSe Li ions , 36. The2/MoC/N–C nanocomposite was synthesized by a temperature-induced method. The interface bridging between MoSe2 and carbon framework was tuned by MoSe2/MoC/N–C connection, greatly improves the structural stability and electronic conductivity and reduces the interfacial charge resistance. Moreover, it also exhibits many features favorable for lithium-ion storage, such as MoSe2 nanodots encapsulated into 3D connected carbon network to improve charge/ion transfer kinetics and minimize the effect of volume expansion; expanded interlayer spacing of MoSe2 to promote lithium-ion diffusion; large electroactive surface area from N–C and rich edge defects of MoSe2; and reinforced structure stability by intermediate in situ MoC. As a result, the as-prepared electrode delivered ultralong and stable cycling performance with a specific capacity of 618 mAh g−1 after 5000 cycles (90% capacity retention) at 2 A g−1. It also shows promising potential in LiFePO4//MoSe2/MoC/N–C full cell (86% capacity retention at 2 A g−1 after 300 cycles). Inspired by the harvest of superior cell performance, in situ engineering corresponding metal carbide as interphase to connect metal dichalcogenide and carbon matrix can be developed as an innovative and effective strategy to achieve high-electrochemical-activity materials.In conclusion, 3D porous MoSeExperimental section, supplementary scheme diagram of interface modification, TG, XRD, BET, SEM, EIS, CV, GITT and equations associated with this article are given in the online version or from the author.Supplementary material 1 (PDF 1435 kb)Below is the link to the electronic supplementary material.