Rhizoctonia solani and promote pepper (Capsicum annuum) growth. Strain SL-44 was isolated from plant rhizosphere and the pot experiment results indicated that the dry and fresh weights of pepper in SL-44 and Rhizoctonia solani (S-R) treatment were 45.5% and 54.2% higher than those in Rhizoctonia solani (R) treatment and 18.2% and 31.8% higher than those in CK  treatment. The plant height in S-R treatment increased by 14.2% and 9.0% compared with those in the R and CK treatments, respectively. In vitro antagonism assay showed that SL-44 exhibited strong antifungal activity against the mycelial growth of Rhizoctonia solani, with an inhibition rate of 42.3%. The amount of phosphorus dissolved by SL-44 reached 60.58 mg·L−1 in broth and 7.5 μg·mL−1 IAA were secreted by SL-44. Strain SL-44 inhibited the growth of R. solani and improved biomass of pepper plants. Mass exchange and information transmission between the pepper plants and SL-44 mutually promoted their development. Bacillus subtilis SL-44 has a great potential as biocontrol agent against Rhizoctonia solani on pepper plants.This study aimed at evaluating the ability of SL-44 to control Pseudomonas, Bacillus, Agrobacterium, Erwinia, Flavobacteria, Pasteuria, Enterobacter, Azospirillum, and Hafnia  with 20 μM EDTA). Briefly, 4 mL aliquot of 1% hydroxypropylmethylcellulose solution was added to the cell suspension. The concentrated root exudates were added to the center of each Petri dish. A ring of turbid zone near the center of each Petri dish appeared after 10–15 min of incubation at room temperature (RT) if the chemotactic response of bacterial cells was triggered.Root exudates were collected according to the root-submerged method proposed by Badri et al.  with somμL of each sample was injected into an Agilent 6890N GC coupled with a 5973 MS to analyze chemical compounds in the root exudates. A DB5-MS (J & W Scientific) 60 m capillary column  was used to achieve the required chromatographic condition, and helium was applied as carrier gas at a flow rate of 1 mL·min−1. The injector temperature was maintained at 250°C, and the temperature program was set as follows: column temperature was held at 50°C for 2 min, ramped at 6°C·min−1 to a final temperature of 250°C, and held for 15 min. The conditions of the mass spectrum included the following: electron impact ion source, EI; ion energy, 70 eV; ionization temperature, 200°C; scanning range, 30–600 amu; and sample volume, 1 μL, with splitless injecting samples.Briefly, 1 P = 0.05).Data were tested for statistical significance using the analysis of variance package included in Microsoft Excel 97, and comparison was done using a least significant difference (LSD) test  at the NCBI website. Analysis using the BLASTn program indicated that SL-44 shared a close relationship as 100% similarity to the DNA sequence of R. solani was examined by dual culture assay in PDA plates. The inhibition zone was clearly observed. SL-44 showed strong antifungal activities against the mycelial growth of R. solani, with an inhibition rate of 42.3% relative to that of CK  . A clearThe root exudates of pepper contain eighteen different organic compounds, namely, seven hydrocarbons, six esters, four aromatic hydrocarbons, and one organic acid . Bacillus subtilis strains had proved its high efficiency on control Rhizoctonia solani, produced IAA, siderophore, and nitrogen fixation to promote plant growth [In many studies,t growth , 25. B. subtilis belong to the same species [ Rhizoctonia solani was inhibited when SL-44 compete with it for nutrients and space on the PDA plate. The inhibition effect of SL-44 could be due to lytic enzymes such as glucanase, chitinase, and protease which were produced, which directly degrade the cell walls of pathogenic fungi, and lipopeptides like iturin and fengycin produced by antagonistic bacteria can be directly against or kill phytopathogens [The physiological and biochemical properties, 16S rRNA sequence analysis, and the high similarity values confirmed that SL-44 and species . Rhizoctathogens , 19, 27. Pseudomonas fluorescens strains L228 and L132 exhibited high potential of P solubilization, with content of soluble P exceeding 1000 mg·L−1; gluconic acid was also produced and detected in the medium as the pH of the medium decreased [ P. putida Rs-198 exhibiting phosphate-solubilizing ability led to varied P concentration ranging from 65 μg·mL−1 to 118 μg·mL−1 in the medium at stationary growth phase. Mardad et al. [ Enterobacter hormaechei subsp. strain PSB 6 that can strongly solubilize P, ranging from 400 mg·L−1 to 505 mg·L−1 after 36 h to 72 h of incubation. The phosphate solubilization ability of SL-44 may promote nutrient uptake and growth of plants.Content of soluble P in SL-44 culture medium was varied during 60 h of incubation. The increase of P-solubilized concentration could be due to the consumption of glucose from the growth media and the release of organic acids, such as gluconic acid, from SL-44. Similar results were extrapolated from a previous study . Pseudomecreased . The redecreased . The abiecreased . The sold et al.  isolated Bacillus subtilis CAS15 reduced the incidence of Fusarium wilt in pepper significantly, by 12.5–56.9%. Sadeghi et al. [ Streptomyces strain C enhanced iron acquisition and wheat growth promotion under saline condition. SL-44 was able to grow in the nitrogen-free culture medium possibly because of the acquisition of atmospheric nitrogen by biological fixation. The changes in the color of the medium could be due to variations in pH when N2 in the air was transformed by the strain into ammonium compounds. The pH of the medium also turned alkaline. In addition, the color of the NFb medium changed to blue [ Klebsiella SN 1.1 reached 790 μg/mL (maximum) at the stationary phase but decreased in the growth medium during the late stationary phase of growth. Shao et al. [ Bacillus amyloliquefaciens SQR9; the IAA concentration reached 9.46 mg·L−1 in the supernatant at stationary phase and decreased in the broth during the late stationary phase of growth (80 h).Siderophore production helps a particular microorganism to compete effectively for available iron against other organisms. The production of siderophore is one of the most common bacterial strategies for acquiring iron under iron-limited conditions. Yu et al.  isolatedi et al.  reported to blue . Hence,  to blue  has scre to blue  reported Bacillus sp. SLS18; the strain produced IAA and siderophores and influenced the biomass production of sweet sorghum (Sorghum bicolor L.). In addition, the nitrogen fixation of SL-44 promoted the nitrogen nutrient absorption of the plant. Taurian et al. [ Pantoea J49 has the ability of increasing the peanut biomass which could be related to mechanisms such as nitrogen fixation and IAA production. Moreover, SL-44 with antagonistic ability in vitro was confirmed to inhibit the mycelium growth of R. solani and directly promote plant growth. Some researchers have found that B. subtilis showed significant inhibitory effects on the growth of R. solani by reduced disease incident and promoted plant growth [Pot experiment showed that SL-44 indeed play a significant role in promoting pepper growth. These findings could be due to the biocontrol properties of SL-44. Hence, SL-44 may improve plant growth and increase plant biomass by several mechanisms, such as production of IAA and siderophore. Luo et al.  determinn et al.  have rept growth , 16.Based on the ability of colonization of SL-44, the results of 0–2 cm colonization can be explained based on root system upgrowth, where the roots become longer and more robust; the bacteria searched for nutrients from deeper roots, leading to decreased colonization with increasing time in S treatment, while the variation of bacterial colonization in S-R treatment may be due to the changed growth condition of SL-44, which takes time to adjust and adapt to the living environment. When the strain adapted to the environment, root exudates and exfoliated root sheath cells can serve as sources of nutrients for microorganisms. SL-44 can exchange information and substances with the roots, thereby colonizing on plant roots stably. However, root exudates and exfoliated root sheath cells cannot be unique nutrients for bacterial growth and reproduction because they cannot satisfy all substances required for the growth of microorganisms. R. solani and SL-44 for spaces and nutrients (S-R treatment). Thus, the number of SL-44 in the roots slightly decreased. Analysis of root colonization within the depths of 0–2 and 2–4 cm showed that although, with the existence of R. solani, SL-44 can compete with them for spaces and nutrients, it also plays an important role in promoting the growth of pepper plant. Hence, SL-44 may protect pepper from R. solani and exhibit high colonization ability. This finding provides a basis for studies on the interaction between strains and plants.The amounts of SL-44 colonization were increased in first 10 days of inoculation with the development of pepper roots at 2–4 cm root length. However, root exudates and exfoliated root sheath cells cannot be unique nutrients for bacterial growth and reproduction because they cannot fulfill entire substances required for the growth of microorganisms. As such, the number of viable SL-44 decreased after 15 days of inoculation. The less colonization in the S-R treatment than that in S treatment might be due to the competition between P. polymyxa SQR-21 and promoted bacterial colonization. Root exudates induce certain beneficial microorganisms to gather and colonize the rhizosphere. Chemotaxis toward root exudates is the first step of bacterial colonization. Previous studies showed that the root exudates of Capsicum mainly contain acids, ketones, amines, esters, alcohols, phenols, hydrocarbons, benzene, and other heterocyclic compounds. Heterocyclic compounds exert many biological activities, such as insecticidal effect, plant growth regulation, and other functions [Bacteria eliciting positive chemotactic effect on a chemotactic attractant will move toward chemotactic substances; as such, the concentration of bacteria in the droplets of chemotactic substances will be higher than the external concentration. Furthermore, a transparent ring was formed at the edge of the droplet . Other sunctions . B. subtilis based on the characterization of its physiology, biochemistry, and 16S rDNA sequence and regarded as potential agent for biocontrol and growth promotion. SL-44 elicited strong inhibitory effect on R. solani. Pot experiment results indicated that SL-44 effectively enhance the biomass and height of pepper seedlings. Moreover, SL-44 exhibited the ability of nitrogen fixation, leading to abundant soluble P and nitrogen that can be absorbed by plants for growth. Furthermore, SL-44 secreted IAA, which can stimulate the growth and development of pepper. The results of colonization and chemotaxis tests showed that SL-44 survived stably in the rhizosphere and interacted with plants. As such, mass exchange and information transmission between the plants and microorganisms mutually promoted their development. Therefore, SL-44 is a suitable agent for biocontrol and plant growth promotion.SL-44 was identified as


Flag leaves (FL) and second leaves (SL) in rice show differential aging patterns during monocarpic senescence. Coordination of aging programs between FL and SL is important for grain yield and quality. However, the molecular bases for differential aging programs between FL and SL have not been systematically explored in rice. Here, we performed mRNA-sequencing of FL and SL at six time points during grain-filling and identified four molecular bases for differential aging programs between FL and SL: phenylpropanoid biosynthesis, photosynthesis, amino acid (AA) transport, and hormone response. Of them, photosynthesis (carbon assimilation) and AA transport (nitrogen remobilization) predominantly occurred in FL and SL, respectively, during grain-filling. Unlike other molecular bases, AA transport showed consistent differential expression patterns between FL and SL in independent samples. Moreover, long-distance AA transporters showed invariant differential expression patterns between FL and SL after panicle removal, which was consistent to invariant differential nitrogen contents between FL and SL after panicle removal. Therefore, our results suggest that the supplies of carbon and nitrogen to seeds is functionally segregated between FL and SL and that long-distance AA transport is an invariant core program for high nitrogen remobilization in SL. Therefore, senescence of leaves greatly affects nutrient supply to grains7. During leaf senescence, photosynthesis (C assimilation) is down-regulated, whereas N remobilization is up-regulated7. Thus, delayed senescence can result in increased grain yields by delaying down-regulation of C assimilation8. On the other hand, delayed senescence can decrease protein content  in grains by delaying up-regulation of N remobilization, whereas accelerated senescence can increase protein content11. Collectively, these data indicate that leaf senescence is crucial for determining both yield and quality of grains during the grain-filling period.In cereal crops, nutrients are predominantly transported to grains from leaves. In rice, leaves provide grains with carbon (C) and nitrogen (N) mainly through assimilation and remobilization, respectivelyOsWRKY4212, OsDOS13, OsTZF114, OsNAP8, and OsNAC10615; (2) hormonal factors, such as the JA receptor CORONATINE INSENSITIVE 1b16; (3) defense-related proteins, such as lesion mimic and early senescence 1 (OsLMES1), spotted leaf3 (OsSPL3), and UDP-N-acetylglucosamine pyrophosphorylase 1 (OsUAP1)19; (4) phytochrome B (OsPhyB)20; and (5) the chlorophyll (Chl) degradation genes 26. Moreover, time-course gene expression profiling of leaves during the grain-filling period can provide a more comprehensive understanding of leaf senescence. Recently, gene expression profiling of flag leaves (FL) from vegetative to senescence stages has identified senescence-associated genes (SAGs), including the six NAC TFs 27.To investigate leaf senescence at the molecular level in rice, several studies have identified a broad spectrum of genes that are associated with leaf senescence using genetic approaches. These include the following: (1) transcription factors (TFs), such as et al.32 analyzed physiological changes, including transitions of Chl, protein, and RNA expression levels, in FL and second leaves (SL), which influence grain-filling the most33. Comparisons of FL and SL showed that FL and SL have differential senescence patterns  during monocarpic senescence and that these differential senescence patterns were altered by panicle removal30. However, FL and SL were shown to perform shared functions to provide C and N to grains33. These data prompted us to characterize the shared and differential senescence programs in FL and SL and to determine the effects of grain-filling on the senescence programs of the two leaf types. Thus, we performed mRNA-sequencing of FL and SL at six time points during the grain-filling period and compared gene expression profiles between FL and SL. These analyses revealed SAGs with shared and differential patterns of expression changes during grain-filling. Mapping of these SAGs into cellular processes identified key cellular processes underlying the shared and differential senescence programs between FL and SL. Furthermore, among differential senescence programs, changes after panicle removal were used to distinguish invariable core senescence programs from variable senescence programs.These studies were performed mostly in FL only. However, senescence in rice does not occur independently in individual leaves and is a systemic process. To characterize this monocarpic senescence, Choudhuri’s Oryza sativa L. ssp. japonica) variety, which has been widely used for genetic analyses34, differential patterns of Chl degradation were observed between FL and SL during grain-filling. Because this is a hallmark feature of leaf senescence, we used this variety for further experiments in this study. To systematically examine temporal characteristics of differential senescence patterns, we initially measured grain weights during the entire grain-filling period. Temporal grain weight patterns revealed that grain-filling was completed at around 32 days after heading (DAH) under our growth conditions  or 36 DAH (OsNAP and OsAAP5 in FL), whereas Chl levels and N contents continuously decreased (see Discussion). Taken together, these data indicate that FL and SL have differential decreases in Chl levels and N contents, which are representative senescence phenotypes during the grain-filling period.In preliminary analyses of the Dongjin  at all six time points), and (2) 3058 genes that showed differential temporal expression patterns between FL and SL during grain-filling   mRNA expression changes in FL 5179 SAGs) and/or SL (4421 SAGs) during grain-filling . To systematically explore the Shared_SAGs, we classified them into seven patterns (SP1-7) , two ABC transporters (Os08g0167000 and Os04g0209200), and a putative copper transporter (Os08g0455900), suggesting that mobilization of molecules using these transporters occurred commonly in both FL and SL during the grain-filling period. Second, DNA methylation/replication/repair, RNA processing, and production of ta-siRNAs/siRNAs involved in RNA interference were represented by SP2 and/or SP3 with early increases during grain-filling. Third, stress responses  were represented by SP4, with gradual increases during grain-filling. Finally, photosynthesis and Chl biosynthetic processes were represented by SP5 and/or SP6, which had early or gradual decreases during grain-filling. These data suggest that DNA repair, epigenetic gene silencing (DNA methylation and siRNA generation), and stress responses were up-regulated with shared temporal patterns in FL and SL during the grain-filling period, whereas photosynthesis-related processes were down-regulated with shared temporal patterns in FL and SL.Several studies have shown that FL and SL perform similar roles to provide nutrients to grainsDifferential decreases of Chl levels and N contents in FL and SL during the grain-filling period Fig.  suggests40, and the pathways for their synthesis are largely shared with the common precursor phenylalanine. These data suggest that protection against stresses involving these molecules increases during grain-filling in both FL and SL, but that the protection capacity is higher in FL than in SL during the middle stage of grain-filling. Second, photosynthetic and Chl biosynthetic processes and lipid and carbohydrate metabolism were represented by G2 . Thus, we focused on the aforementioned GOBPs represented by G1-4. These data suggest four potential molecular bases (Bases 1-4) for the differential mRNA expression patterns in FL and SL during the grain-filling period Fig.  as folloThe above analyses show that several cellular processes are represented by both Shared_SAGs and Diff_SAGs as follows: (1) The transport process by SP1 of Shared_SAGs 41, the adaptor component (OsLhcB5), and the assembly/repair component (OsPsb28)42 , which are conserved or specifically present in higher plants43  were observed during grain-filling , photosystem II (PS II), cytochrome bsCO Fig. . Among t)42 Fig. . In conts43 Fig. . Howevering Fig. . CollectOsGluTR), protoporphyrinogen oxidase (OsPPOX), two subunits of magnesium-chelatase (OsChlH/I)44, and a proteinaceous cofactor genome-uncoupled 4 (OsGUN4)45, showed differential down-regulation patterns in FL and SL. These enzymes are key to the direction of metabolic flux into Chl biosynthesis at branching points in the metabolic pathway  and protochlorophyllide reductases A and B (OsPORA and OsPORB), which catalyze several intermediate reactions in the pathway, showed shared down-regulation patterns in FL and SL. Collectively, these data suggest that Chl biosynthetic pathway activities showed largely differential down-regulation patterns in FL and SL, resulting in higher Chl levels in FL  and SP5-6 (Shared_SAGs) into the Chl biosynthetic pathway Fig. . Five enway Fig. . In cont FL Fig. . Howeveret al.30 previously showed that panicle removal changes senescence patterns of FL and SL from non-sequential to sequential. Thus, we investigated the relative effects of grain-filling on senescence of FL and SL in panicle removal experiments and measured temporal expression patterns of the following representative genes , phenylalanine ammonia-lyase (OsPAL), and flavonoid 3′-monooxygenase (OsF3′H) for phenylpropanoid biosynthesis (Basis 1 for G1); (2) light harvesting complex Chl a/b binding protein (OsLhcB1), glutamyl-tRNA reductase (OsGluTR), and magnesium-chelatase subunit H (OsChlH) for photosynthesis and Chl biosynthesis (Basis 2 for G2); (3) UMAMIT19 homolog (OsUMAMIT19), OsATL9, and OsCAT4 for AA transport (Basis 3 for G3); and (4) OsERF75, OsERF77, and OsERF96 for hormone responses (Basis 4 for G4). In addition to panicle removal experiments, we confirmed differential expression patterns of these representative Diff_SAGs in mRNA sequencing experiments  may act as an invariant core differential senescence program between FL and SL, which should be robustly achieved under environmental variations during the grain-filling period. In contrast, the other bases likely serve as variable differential senescence programs between FL and SL. N is an essential nutrient for plant growth and seed development, and stable N supply to grains provides N for germination and seedling growth. Accordingly, N supply during the grain-filling period can be predominantly achieved by AA transport from leaves to grains.46, 62 were expressed in FL or SL during grain-filling , G3 (6 AATs), and G5 (1 AAT). G1 showed higher expression in FL than in SL, G5 showed down-regulation during grain-filling in SL, and G3 showed up-regulation during grain-filling. These data indicate increased AA transporting activities in SL, suggesting greater relevance to lower total N content in SL than in FL  were also invariant after panicle removal. We measured expression levels of these genes with and without panicle removal at the six time points during grain-filling. OsAAP5/8 and OsBAT4 were excluded from this analysis because expression levels of OsAAP8 were too low for reliable measurement and in contrast with mRNA-sequencing data, OsAAP5 and OsBAT4 failed to show differential expression patterns between FL and SL in independent cohorts  that belonged to neither G3 nor the three long-distance AAT subfamilies  in G3 were altered to a smaller degree in the differential expression patterns between FL and SL , and stress responses, whereas Diff_SAGs were mainly associated with phenylpropanoid biosynthesis (Basis 1), PS II-based photosynthetic and Chl biosynthetic processes (Basis 2), transport of AAs (Basis 3), and senescence-associated hormone responses (Basis 4). These data indicated that Shared_SAGs and Diff_SAGs mediate shared and differential molecular aging patterns between FL and SL during grain-filling.Leaf senescence significantly affects yield and quality of grains54. According to our analyses of Bases 1–2 of the Diff_SAGs, FL showed higher expression levels of genes involved in photosynthesis  or N remobilization (OsNAP8 and OsAAP537) during grain-filling. In contrast with previous studies that report expression levels of these genes up to 28 DAH8, we continued to measure their expression levels up to 44 DAH and showed peaks in temporal expression patterns at 28 DAH. In agreement with our data, the RiceXPro56 database includes the time-course gene expression profiles up to 35 DAH and shows that OsSGR1, OsPPH1, OsRCCR1, and OsNAP expression levels peak at 28 DAH  can vary depending on the cutoffs used to identify the Diff_SAGs. Thus, we applied the following four additional stringent criteria to identify Diff_SAGs: Criteria 2–3) log2-fold-changes ≥0.58 and P < 0.05 from t-test in at least two (Criteria 2) or three (Criteria 3) of the six time points; and Criteria 4–5) log2-fold-changes ≥1 and P < 0.05 in at least one (Criteria 4) or two (Criteria 5) of the six time points. Using Criteria 1–5, 3058, 1934, 1185, 960, and 518 Diff_SAGs were identified, respectively  represented by the Diff_SAGs identified using Criteria 1–4. In contrast, the GOBP (defense response) for G5 was not significantly represented by the Diff_SAGs identified using Criteria 2–5 while the GOBP (photosynthesis) for G2 was not significantly represented by the Diff_SAGs identified using the most stringent criteria (Criteria 5). These data suggest that defense responses for G5 are sensitive to differing cutoffs, but that the GOBPs for G1–4 are relatively robust. Moreover, the GOBPs for G1–4 can vary depending on the tool used to identify the Diff_SAGs. Thus, we applied the timecourse Package developed by Tei and Speed57 to our data and identified 2136 Diff_SAGs . Among these, 969 (45.37%) overlapped with our Diff_SAGs, and 336, 180, 186, 248, and 132 belonged to G1-5, respectively. The GOBP enrichment analysis for these genes in G1-5 showed that the GOBPs for G1–4 were significantly represented by the corresponding Diff_SAGs  based on the GOBPs represented by the Diff_SAGs in G1–4 identified using the following criteria (Criteria 1): log60; and (2) photosynthetic activity is reduced when nutrient sinks are removed and the need for C assimilation decreases65. Similarly, the bases representing phenylpropanoid biosynthesis (Basis 1) and senescence-associated hormone responses (Basis 4) are reportedly sensitive to various environmental factors: (1) flavonoids can be induced by UV-B66; and (2) amounts of senescence-associated hormones, such as abscisic acid, ethylene, and jasmonic acid, can be varied in response to various types of stimuli67. Unlike the variable bases, however, no previous reports have shown that AA transport (Basis 3), particularly long-distance AA transport  competition between C and N for captured energy during grain development72 and (2) N dilution by C-based compounds73. Based on the present observations of variable and invariant bases, we suggest that the negative relationship between grain yield and protein content reflects differential sensitivity of photosynthesis (variable Basis 2) and AA transport (invariant Basis 3) to senescence-inducing stresses, such as drought, salinity, and diseases or pests74. Specifically, photosynthetic activity was sensitively down-regulated during stress-induced senescence, resulting in reduced grain yield. In contrast, AA transport activity was relatively stable, leading to stable protein contents in grains. Given the stable protein content, under stress conditions, photosynthetic activity can be further decreased, resulting in a yield relatively lower than can be explained by protein content in grains. Under non-stress conditions, however, photosynthetic activity can be less decreased, resulting in a yield relatively higher than indicated by protein contents.Variable and invariant bases of differential expression patterns between FL and SL likely reflect frequently observed negative genetic relationships between grain yields and grain protein contents in crops, such as soybean, wheat, maize, and riceVariable and invariant components are often observed in biological systems and in physical and chemical systems. These systems have a general structure wherein invariant components are located at the core and variable components are peripheral and interact with core components and external factors. For example, circadian clocks have the core clock components (LHY and TOC1), which are relatively stable under environmental variations. These core clock components constantly interact with variable components, including CCA1, ELFs, PRRs, GI, ZTL, and PIFs, at outer layers, which respond to changes in various environmental factors. Thus, the transcriptional programs for the regulation of differential aging in FL and SL likely form a similar structure to ensure the stability of core components, such as stable protein contents in grains through differential long-distance AA transport for N remobilization from SL, and to cope with environmental changes through variable bases  photosynthesis and plant growth are down-regulated until favorable conditions are recovered, and (2) when the need for C in newly developing sinks increases, photosynthesis increases in source leaves. Furthermore, analyses of other systems indicate close interactions of the core basis (AA transport) with variable bases.78. Hence, invariant supplies of AAs can also facilitate seed germination even in N limiting soil. Taken together, these data suggest that the stable supply of AAs to grains is of high priority and is met by differential aging regulation between FL and SL. Concomitantly, variable bases accommodate environmental variations. Therefore, transgenic rice or farming practices that enhance long-distance AA transport in SL can be developed to maximize the benefits of differential aging regulation between FL and SL.Finally, our findings can be useful in developing transgenic plants and farming practices  that optimize rice grain yields and protein contents under given conditions. Our results suggest that leaves ensure first the limiting N resource in grains through differential long-distance transport of AAs between FL and SL regardless of environmental conditions. Moreover, invariant supplies of AAs to grains likely maintain minimal plant growth when photosynthesis is severely blocked, because AAs can be used as the C source. For example, glutamate dehydrogenase produces the TCA cycle intermediate 2-oxoglutarate from glutamateOryza sativa L. ssp. Japonica) for mRNA-sequencing were transplanted in a paddy field at Daegu, South Korea (approximately 35 °N) in early June in 2013. Seedlings for qRT-PCR analyses  were transplanted in a different field at the same time in 2014. In both years, heading dates were around August 20, and stems with similar heading dates were marked for sample preparation and physiological analyses. Samples were prepared between 7 and 8 AM at six time points . Middle parts16 of nine independent leaves were obtained as a biological replicate and three replicates were sampled at the same time.Rice seedlings of the Asian rice cultivar Dongjin . RNA integrity was confirmed using an Agilent Technologies 2100 BioAnalyzer (RNA integrity number >8.0). Poly(A) mRNA was then isolated from total RNA, and fragmentation reactions were performed using Illumina Truseq Stranded mRNA LT Sample Prep Kit with poly-T oligo-attached magnetic beads according to the manufacturer’s instructions. Reverse transcription of RNA fragments was performed using Superscript II reverse transcriptase . Adaptor-ligated libraries were generated for the resulting samples and were then sequenced using Illumina HiSeq 2500. Paired-end sequencing was performed to 101 bp from both ends for three independent replicates at each time point under individual conditions.79 (version 1.2.1) with the adapter sequences . Read alignment was performed using TopHat aligner80  with two mismatches and up to 10 multiple alignments allowed based on Oryza sativa reference genome (Oryza sativa IRGSP-1.0.22). We then assembled the aligned reads into transcripts based on gene features (GTF file of Oryza sativa IRGSP-1.0.22. gtf) and used Cufflinks81  to calculate the fragments per kilobase per million mapped reads (FPKM) values for the assembled transcripts. Quantitated transcripts whose FPKMs are larger than 1 in at least one time point were regarded as expressed transcripts as previously described82. For the statistical test and pattern analysis of the transcripts, we obtained the aligned read counts using HTSeq.83 (version 0.6.1) based on gene features and then performed a two-stage normalization as previously described85. Briefly, we first normalized the aligned read counts into count per million (CPM) using The trimmed mean of M-values (TMM) normalization method86 in edgeR package87 to remove the bias of RNA composition and the difference of library sizes across the samples and then performed the quantile normalization88 on log2(CPM + 1) to remove systematic variation in the CPM values across the samples. Note that one was added to CPM before taking the log to avoid negative infinity.For the read sequences obtained from mRNA-sequencing, adapter trimming was performed using cutadapt software2-CPM values were divided into time-course expression profiles of FL and SL. The time-course expression profiles of FL or SL were smoothed using the Savitzky-Golay method89 with polynomial order = 2 and frame size = 5. For each transcript, we calculated the Pearson correlation coefficients (PCCs) between the smoothed expression profiles from three possible pairwise combinations of the three biological replicates . The transcripts with the median PCCs ≥0.7 were selected as the ones with reproducible age-dependent expression patterns. The cutoff of 0.7 was determined from the following random experiments: (1) for n × m time-course mRNA expression data matrix (X) for n transcripts and m time points in each replicate of FL or SL, we randomly permuted the columns of X; (2) for each transcript, we calculated PCC between temporal expression profiles using the randomly permuted data matrixes (Xrand) of the replicates; (3) steps 1 and 2 were repeated 100 times; and (4) we estimated an empirical distribution for the null hypothesis  using the PCCs obtained from the random permutations. The 95th percentile of PCC in the empirical null distribution was 0.71. Based on this finding, we determined the PCC cutoff to be 0.7. The distribution of the number of transcripts with respect to PCC with an interval of 0.7 from zero to one revealed that for FL, there were 17696 (46.5%) with significant (P < 0.05) correlations (PCC ≥ 0.7), 4079 (10.7%) with marginal correlations (0.4 ≤ PCC ≤ 0.7), and 8945 (23.5%) with low correlations (PCC < 0.4), and for SL, there were 13681 (36.0%) with significant (P < 0.05) correlations with (PCC ≥ 0.7), 5671 (14.9%) with marginal correlations (0.4 ≤ PCC ≤ 0.7), and 10956 (28.8%) with low correlations (PCC < 0.4). For FL, we selected the transcripts with the median PCC ≥ 0.7 based on the time-course expression profiles of FL and then identified FL_SAGs as the selected transcripts with P < 0.05 (ANOVA with Bonferroni correction as a post-hoc test) and the maximum absolute log2-fold-changes ≥1 over six time points. Using the same approach, we selected the transcripts with the median PCCs ≥0.7 based on the time-course expression profiles of SL, and then identified SL_SAGs. Finally, we defined overall SAGs as the union of FL_SAGs and SL_SAGs. For the overall SAGs, log2-fold-changes were calculated by subtracting the average log2-CPM values at 4 DAH in FL from those at individual time points. These log2-fold-changes for FL and SL were displayed in Fig. The normalized log2-fold-changes between FL and SL were calculated by subtracting the median log2-CPM values of the three replicates of SL from those of FL at individual time points. Of the overall SAGs, Shared_SAGs were identified as the SAGs with log2-fold-changes <0.58 (1.5-fold) for all six time points. We also applied the two-sample t-test to log2-CPM values of the three replicates of FL and SL at each time point to evaluate the significance  of the difference in expression levels between FL and SL at the corresponding time point. Of the overall SAGs, Diff_SAGs were then identified as the SAGs with P-values < 0.05 from the t-test and absolute log2-fold-changes ≥0.58 (1.5-fold) in one or more of the six time points. Moreover, to examine whether the method used to identify Diff_SAGs can affects the GOBPs represented by Diff_SAGs, we also identified Diff_SAGs by applying the timecourse Package developed by Tei and Speed57 to our data. For each gene, the Package provides no P-values, but only Hotelling T2 statistic values representing the significance of the difference between its temporal expression profiles of FL and SL. Thus, to estimate P-values for the Hotelling T2 values, we performed the following random permutation experiments: (1) for n × m time-course mRNA expression data matrix (X) for n genes and m time points in each replicate of FL or SL, we randomly permuted the columns of X; (2) for each gene, we calculated a Hotelling T2 value by applying the timecourse Package to the randomly permuted data matrixes (Xrand) of the replicates of FL and SL; (3) steps 1 and 2 were repeated 1000 times; (4) we estimated an empirical distribution for the null hypothesis  using Hotelling T2 values obtained from the random permutations; and (5) for the Hotelling T2 value of each gene, we calculated an adjusted P-value by one-sided test using the empirical distribution. We then selected 2136 Diff_SAGs with P ≤ 0.05 and of these, 336, 180, 186, 248, and 132 belonged to G1-5, respectively.For each of the aforementioned overall SAGs, log2-fold-change profiles by the k-means clustering method with correlation as a similarity measure and the number of clusters (k) = 30. The 30 clusters (SC1-30) of Shared_SAGs were categorized into 16 up-regulated and 14 down-regulated clusters. The up- and down-regulated clusters were further grouped into SP1–4 and SP5-7, respectively, by applying hierarchical clustering (complete linkage method and Euclidean dissimilarity measure) to the average log2-fold-change profiles of the up- and down-regulated clusters. Also, Diff_SAGs were clustered using their log2-fold-change profiles and also additionally the differences of log2-fold-changes between FL and SL by the same k-means clustering method. The 30 clusters (DC1-30) of Diff_SAGs were then categorized into 15 up-regulated and 15 down-regulated clusters. The up- and down-regulated clusters were grouped into DP1-10 and DP11–16, respectively, by applying the same hierarchical clustering used for the Shared SAGs. Finally, DP1–16 were further categorized into five major groups (G1-5) based on the similarity in the difference patterns of log2-fold-changes between FL and SL.Shared_SAGs were clustered using their logOryza sativa were collected from gramene (release 41)90, NSF45K annotation91, and RGAP all GO slim92. GOBPs enriched by a group of the genes in SP1-7 and G1-5 were identified using curated GOBP annotations by the EASE scoring method used by DAVID software93. For each group, we selected the GOBPs with P-values ≤ 0.05 and the numbers of genes with the GOBPs ≥5.GOBP annotations of After DNase AM1906, ambion) treatment, cDNA was synthesized from approximately 400 ng of total RNA using an ImProm-II Reverse Transcription System . Subsequently, qRT-PCR analyses were performed using CFX96 or CFX384 Touch™ Real-Time PCR Detection Systems (Bio-Rad) with cDNA from 10–15 ng of total RNA. Actin mRNA expression was used as an internal standard, and normalized cycle threshold (Ct) values were used in statistical analyses Con] was then calculated by integrating the differences of the mean expression levels at individual time points over time using the trapezoidal integration method. [Diff(FL-SL)PR − Diff(FL-SL)Con]/Diff(FL-SL)Con were calculated. The same method was used for long-distance AATs and the negative control AATs.Mean relative expression levels of three replicates in FL and SL measured from qRT-PCR analysis were first computed at individual time points, and the differences between the mean relative expression levels of FL and SL were then calculated at individual time points. The integrated difference with [Diff(FL-SL)2-CPM values from mRNA-sequencing and the expression levels from qRT-PCR in FL or SL were smoothed using the Savitzky-Golay method with polynomial order = 2 and frame size = 5 as previously described82. The correlation coefficients between the smoothed mRNA-sequencing and qRT-PCR data were calculated using three different methods . In each method, the correlation coefficients for FL and SL were weighted summed. For mRNA-sequencing or qRT-PCR data, the weights for FL and SL were calculated as the areas under the smoothed expression profiles of FL and SL, respectively. For FL or SL, the final weight was then computed as the geometric mean of the weights of the corresponding leaf for mRNA-sequencing and qRT-PCR data. The weighted summed correlation coefficients from the three methods were finally combined into their geometric mean.For each of the representative genes for G1-5, log94. Ground leaf tissues were then dried and total N contents were measured from approximately 2 mg tissue samples using an Elemental analyzer (Elementar).Chls were extracted from approximately 50 mg of ground tissue samples using 95% ethanol. Chl contents were calculated after measuring optical densities using a 96 well plate reader 95 with the accession number GSE89233.The mRNA sequencing data were deposited into the Gene Expression OmnibusSupplementary informationSupplementary dataset


We firstly give a modification of the known Hermite-Hadamard type inequalities for the generalized k-fractional integral operators of a function with respect to another function. We secondly establish several Hermite-Hadamard type inequalities for the generalized k-fractional integral operators of a function with respect to another function. The results presented here, being very general, are pointed out to be specialized to yield some known results. Relevant connections of the various results presented here with those involving relatively simple fractional integral operators are also indicated. I is an interval in the real line A function I is an interval) is a convex function and e.g., [One of the best-known inequalities for convex functions is the following Hermite-Hadamard inequality: If entclass1pt{miniman .α follow easily by setting It is easy to verify that t}w=0 in  and 9)..\documenet al. [Budak et al.  establisLetbe a differentiable mapping onwithandThene.g., [f with respect to the function g on α are defined, respectively, byf by a function g of the order α. Choosing e.g., [We recall the following generalized fractional integral operators =x in  and 4),,e.g., [\mentclasspt{miniman \documentperators  and 9),,\document}w=0 in  and (21)et al. [The Hermite-Hadamard type inequalities in  have beeet al.  who usedLetandbe a bounded sequence. Also, letbe an increasing and positive function onhaving a continuous derivativeonIffis a convex function onthen the following Hermite-Hadamard type inequalities for the generalizedk-fractional integrals offwith respect to the functiongonin Let\docued as in .I be an interval, such that We begin by recalling some notations given in . Let I bTaking t}s=1 in  and 32)\documentThe Hermite-Hadamard type inequalities for the generalized k-fractional integrals of a function with respect to another function in Theorem Letandbe a bounded sequence. Also, letbe an increasing and positive function onhaving a continuous derivativeonIffis a convex function onthen the following Hermite-Hadamard type inequalities for the generalizedk-fractional integrals offwith respect to the functiongonin Let\docued as in .f is convex on Since s, with the aid of , , , , 39), we hality in .f on s, similar to the proof of the first inequality, we haves, we obtainTo prove the second inequality in , using tAdding  and 42)42), we hality in . Hence tSetting entclass1pt{minimaLetandbe a bounded sequence. Also, letbe an increasing and positive function onhaving a continuous derivativeonIffis a convex function onthen the following Hermite-Hadamard type inequalities for the generalized fractional integrals offwith respect to the functiongonin Let\docued as in .Further, choosing mentclass2pt{minimLetandbe an increasing and positive function onhaving a continuous derivativeonIffis a convex function onthen the following Hermite-Hadamard type inequalities for the generalized fractional integrals offwith respect to the functiongonin Let\docued as in .It is remarked in passing that choosing mentclass2pt{minimWe begin by presenting an integral formula involving the functions  and 32)32), whicLetandbe a bounded sequence. Also, letbe an increasing and positive function onhaving a continuous derivativeonFurther, letbe a differentiable mapping onandThenwhereandare given as in (and (en as in  and 32)Let\docuUsing  and chanSimilarly, using  and chanUsing  to add  and 49)49), we oapplying -34) to  to 50\doapplying . □Setting entclass1pt{minimaLetandbe a bounded sequence. Also, letbe an increasing and positive function onhaving a continuous derivativeonFurther, letbe a differentiable mapping onandThenwhereandare given as in (and (en as in  and 32)Let\docuChoosing entclass1pt{minimaLetwithAlso, letbe an increasing and positive function onhaving a continuous derivativeonFurther, letbe a differentiable mapping onandThenwhereandare given in (and (given in  and 15)Let\docuSetting entclass1pt{minimaTaking entclass1pt{minimaLetandbe a bounded sequence. Also, letbe an increasing and positive function onhaving a continuous derivativeonFurther, letbe a differentiable mapping onsuch thatandis a convex function onThenwhere the notations are given as above andg is increasing on Here and in the following, let ality in . Since g view of , 31) an an\docum view of , we findentclass1pt{minima|, using  and 30)\documentChoosing Letandbe an increasing and positive function onhaving a continuous derivativeonAlso, letbe a differentiable mapping onsuch thatandis a convex function onThenwhereandSetting The special case of Theorem t}q=1 in , TheoremLetandbe a bounded sequence. Also, letbe an increasing and positive function onhaving a continuous derivativeonFurther, letbe a differentiable mapping onsuch thatis convex forwithThenwhere the notations are given as above andUsing convexity of entclass1pt{minimaSetting Letandbe an increasing and positive function onhaving a continuous derivativeonAlso, letbe a differentiable mapping onsuch thatis convex forwithThenwhereis given in (andgiven in ,\documeSetting Choosing Letandbe a bounded sequence. Also, letbe an increasing and positive function onhaving a continuous derivativeonFurther, letbe a differentiable mapping onsuch thatis convex forThenwhere the notations are given above:andUsing convexity of entclass1pt{minimaSetting Choosing Setting


Mecp2308/y mice exhibited reduced heroin seeking behavior in CPP test. These data reveal a functional role of miR-218 and its target, MeCP2, in the regulation of heroin-induced behavioral plasticity.MicroRNAs (miRNAs) are a class of evolutionarily conserved, 18–25 nucleotide non-coding sequences that post-transcriptionally regulate gene expression. Recent studies implicated their roles in the regulation of neuronal functions, such as learning, cognition and memory formation. Here we report that miR-218 inhibits heroin-induced behavioral plasticity. First, network propagation-based method was used to predict candidate miRNAs that played potential key roles in regulating drug addiction-related genes. Microarray screening was also carried out to identify miRNAs responding to chronic heroin administration in the nucleus accumbens (NAc). Among the collapsed miRNAs, top-ranked miR-218 was decreased after chronic exposure to heroin. Lentiviral overexpression of miR-218 in NAc could inhibit heroin-induced reinforcement in both conditioned place preference (CPP) test and heroin self-administration experiments. Luciferase activity assay indicated that miR-218 could regulate 3′ untranslated regions (3′ UTR) of multiple neuroplasticity-related genes and directly target methyl CpG binding protein 2 (Mecp2). Consistently,  Drug addiction is a psychiatric disorder characterized by loss of control over drug consumption and compulsive drug taking despite serious negative consequences456Recent studies indicate that epigenetic regulation of gene expression plays an important role in neurogenesis, synaptic plasticity and neurological disorders891112151617In this study, we utilized bioinformatic approaches to predict potential key regulators in drug addiction. Among the top-ranked miRNAs, we found that miR-218 is down-regulated in response to chronic heroin administration. Lentiviral-mediated miR-218 overexpression significantly attenuated heroin-induced reinforcement in both conditioned place preference (CPP) and self-administration (SA) model. These effects were proposed to be mediated by suppression of target genes such as Mecp2, that participates in epigenetic control of gene transcription. Our observation provides a possible miRNA-mediated epigenetic regulatory mechanism in heroin addiction.etc. were top-ranked and previously reported172021First, we tried to screen for miRNAs involved in drug addiction-related regulatory networks. A miRNA could regulate gene expression either by directly targeting drug addiction-related genes, or by targeting regulatory elements (e.g. transcription factors) whose impact may propagate across the whole regulatory network. Thus we utilized a network propagation-based model (NP-method)Microarray screening was carried out to identify miRNAs responding to chronic heroin administration. As shown in Among these 16 candidate miRNAs, miR-181a, a miRNA previously implicated in cocaine-induced plasticity = 11.80, p = 0.003). Moreover, Bonferroni’s post hoc analysis revealed that rats with miR-218 overexpression in the NAc exhibited reduced preference to the heroin-paired chamber, compared with LV-control injected rats  or scramble control sequence (LV-control) into the NAc region . The expted rats .Next, we carried out heroin self-administration tests to further validate the role of miR-218 in heroin seeking behavior. Rats were trained to self-administer heroin for 8 consecutive days, then subjected to LV-miR-218 or LV-control injection. Consistent with CPP results, miR-218 overexpression significantly reduced heroin acquisition compared with the control group .Renilla luciferase and co-transfected the constructs with miR-218 mimic or scramble control in HEK293T cells. Reduced luciferase activity indicated that these genes were the directly regulated by miR-218 In the present study, we found that miR-218 was downregulated by chronic heroin use in NAc. Consistently, bioinformatic analysis indicated the predicted targets of miR-218 was enriched in addiction-related genes and involved in neuroplasticity. MiR-218 is encoded by an intron of the Slit gene and inhibits the expression of Robo1 which play important roles in axonal growth3638Mecp2308/y mice. However, Deng et al. found that Mecp2308/y mutant mice showed enhanced locomotor response to acute amphetamine administration and impaired CPP, but lentivirus mediated knockdown or overexpression of MeCP2 in NAc enhanced or inhibited amphetamine-induced locomotion and CPP respectivelyMecp2 Ser421Ala knock-in mice display both a reduced threshold for amphetamine sensitization and enhanced behavioral sensitivity to the reinforcing properties in cocaine self-administration, indicating that MeCP2 in NAc limited behavioral plasticity upon repeated psychostimulant exposure3050MeCP2 is a transcription factor binding to methylated cytosine residues in DNA and recruits histone deacetylases and other transcriptional repressors to silence target genes4546Taken together, our results reveal that miR-218 is involved in heroin induced behavioral plasticity possibly by regulating Mecp2. Overexpression of miR-218 in NAc decreased heroin conditioned preference and heroin self-administration. MiR-218 is likely to contribute to heroin related modifications, both transcriptionally and epigenetically. Our results provide a promising and novel approach to the treatment of heroin addiction. However, the role of miR-218 in other drug induced plasticity and the downstream signaling pathways regulated by miR-218 in addiction are remained to be elucidated.Mecp2308/y mice and littermates were maintained by mating heterozygotes with C57BL/6 J. All animal treatments were in strict accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by Animal Care and Use Committee of Shanghai Medical College of Fudan University.Male Sprague-Dawley rats were purchased from the Shanghai SLAC Laboratory Animal Co. Ltd. and housed with a 12 h reverse dark/light cycle at 23 °C. All subjects were allowed free access to food and water. Rats used for experiments were 8–10 weeks old. 8 IU/ml.The lentivirus construct and packaging were performed as we described previouslyTo facilitate subsequent heroin self-administration, rats were trained to press an active lever for food pellets until they self-administered 100 pellets for 3 consecutive days on fixed-ratio 1 program. Then the rats were anesthetized, and a silastic catheter (0.04 cm interior diameter) was inserted 3 cm into the jugular vein, and the other end of the catheter was attached to a stainless steel pedestal and mounted on the skull. Bilateral 26-gauge guide cannulae were implanted in NAc . After surgery, the rats were allowed to recover for 7 days and the catheters were flushed daily with saline supplemented with heparin (30 IU/ml) and gentamycin (5 mg/ml). Next the rats were allowed to self-administer heroin  during a daily 4 h session. After the 8th session, the rats were anesthetized and subjected to lentivirus infusion with needle  that extend 1.5 mm beyond the guide cannulae. The self-administration procedure was resumed 3 days after lentivirus injection and tested on FR1 schedule for six consecutive days.Mecp2308/y and Mecp2+/y mice followed the same procedure.The apparatus was three-chambered (Med Associates), with two large side chambers with distinct floors and decorations and one small center chamber. In pretest, the rat was first confined in the center chamber for adaption for 5 min, and then allowed to freely explore the chambers for 15 min. Unbiased rats were subjected to lentiviral injection into NAc . The rats were allowed to recover for 7 days. Then the rats were alternatively confined in each large chamber for 30 min after injection of heroin  or saline . On the test session (day 8), the rats were adapted in center chamber for 5 min and allowed to explore all chambers unrestrictedly for 15 min. The time which the rat spent in each large chamber was recorded and CPP score was defined as the time spent in the heroin-paired chamber minus the time spent in the saline-paired chamber during the CPP test. The CPP pretest, conditioning and test procedure of Total RNA are harvested using TRIzol (Invitrogen) and RNeasy mini kit (QIAGEN) according to manufacturer’s instructions. The RNA samples are labeled using the miRCURY™ Hy3™/Hy5™ Power labeling kit and hybridized on the miRCURY™ LNA Array (v.11.0). Scanning was performed with the Axon GenePix 4000B microarray scanner. GenePix pro V6.0 is used to read the raw intensity of the image. Median normalized signal of each miRNA was used to represent expression level. MiRNAs that exhibit 1.25-fold up-regulation or 0.75-fold down-regulation was chosen for further analysis.Network propagation based model was generated as described−ΔΔCt method.TaqMan small RNA assay (Applied Biosystems) for miRNAs of interest were performed. U6 was used for normalization. Primers used in RT-PCR were Mecp2 , Gabrb3 , GluR2 , Dnmt3a , Ube3a , Nrxn1 , Sema6b , Gng3 , β-actin . All samples were run in duplicate. The relative expression level of mRNA was calculated by 2Renilla luciferase of psiCHECK-2 vector. The target sequences were, Gabrb3 [CCTTTATTTCTGTACTAACTTATCTCATAAGCACACCCAATTCCTCCTAG], GluR2 [TATTGTTAGTCTCTTGATTCATAATGACTTAAGCACACTTGACATCAACT], Dnmt3a [TTGGTTGTCTCTAGCCTGATCAGATAGGAGCACAAACAGGAACAGAATAG], Ube3a [TCTTTGTAGCTGGACAGCACAATGTTTATGATTTATTTAATCTGTAGTTT], Nrxn1 [AAACTTATTTACTTTCCTTTTTATGAAGCACATACAAAAGAAGACAGGGA], Sema6b [CGGGTGGGGATCTCCTCGCCACAGGGAAGCACAAGAGCCCCCTCCATCCC], Gng3 [CGCACTTATCCTGAGATTATCTGAAGCACAAGGCCCTCCTTACCCACCTC]. Mecp2 [TTGGGATGTTTTTCTTACCGACAAGCACAGTCAGGTTGAAGACCTAACCA]. We transfected 500 ng recombinant vector with 10 pmol miRNA mimic or scramble  into HEK293T cells with Lipofectamine 2000 (Invitrogen). After 24 h, dual-luciferase report assay system (Promega) was performed to measure Renilla and Firefly luciferase activity, then Renilla activity was normalized to the Firefly luciferase activity. All the experiments were repeated for at least three times.Predicted target sequences of miR-218 were cloned to the 3′ UTR of The rats were anesthetized and transcardially perfused with saline, followed by 4% paraformaldehyde in PBS. Then the brains were quickly removed and post fixed with 4% paraformaldehyde at 4 °C for about 4 h and stored in 30% PBS-buffered sucrose solution for 72 h. The brains were sectioned (40 μm) with a cryostat (Leica) and the sections were washed in PBS, blocked with blocking buffer (10% donkey serum in PBS containing 0.3% Triton X-100) for 1 h, then incubated with NeuN antibody  at 4 °C overnight. Sections were subsequently rinsed with PBS and incubated for 1 h at room temperature with CY3 goat anti-rabbit antibody . The images were captured with a microscopic CDD camera (Olympus).t-test or one-way analysis of variance (ANOVA). For all the behavior results, data were analyzed by two-way repeated measures ANOVA followed by Bonferroni’s post hoc test. Luciferase activity data were analyzed by student’s t-test or two-way ANOVA followed by Bonferroni’s post hoc test. P < 0.05 are defined as statistically significant.Data are presented as mean ± s.e.m. For quantitative PCR results, data were analyzed by student’s How to cite this article: Yan, B. et al. MiR-218 targets MeCP2 and inhibits heroin seeking behavior. Sci. Rep.7, 40413; doi: 10.1038/srep40413 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


This study evaluates the fracture properties and rear-face strain distribution of nonreinforced and hooked steel fiber-reinforced concrete panels penetrated by projectiles of three different nose shapes: sharp, hemispherical, and flat. The sharp projectile nose resulted in a deeper penetration because of the concentration of the impact force. Conversely, the flat projectile nose resulted in shallower penetrations. The penetration based on different projectile nose shapes is directly related to the impact force transmitted to the rear face. Scabbing can be more accurately predicted by the tensile strain on the rear face of concrete due to the projectile nose shape. The tensile strain on the rear face of the concrete was reduced by the hooked steel fiber reinforcement because the hooked steel fiber absorbed some of the impact stress transmitted to the rear face of the concrete. Consequently, the strain behavior on the rear face of concrete according to the projectile nose shape was confirmed. Front-side failure is obtained when a projectile strikes concrete. At this point, a compressive stress wave is generated and radially transmitted to the rear face of the concrete. This compressive stress wave is reflected as a tensile stress wave when it reaches the rear face. This action causes a decrease in the magnitude of the compressive wave and an increase in the magnitude of the tensile wave. Cracks and fractures will occur if the generated tensile stress exceeds the dynamic tensile strength of the concrete and the strain limits at any point in the concrete matrix  1]. Pri. Pri1]. The suppression of a missile perforation failure is generally considered a priority in the protective design of military facilities. In civilian applications, maximizing human safety and minimizing property damage in the event of concrete scabbing are a priority for general infrastructure design. Therefore, a concrete specimen thickness that can prevent scabbing and perforation by projectile impact is considered fundamental in protection design.Various empirical formulae have been proposed by researchers to predict the penetration depth, scabbing limit thickness, and perforation limit thickness of concrete based on various impact conditions. The application ranges of these empirical formulae are limited by the data ranges associated with their derivation. Regression techniques were applied to the development of equations for calculating the scabbing and perforation limit thicknesses based on the penetration depth ,12. The Expanded applications include considering different projectile nose shapes  in predicting local fracture. Several researchers applied nose shape considerations to the penetration depth calculations. Almusallam et al.  used a bFocusing more on formulation, Wen et al.  proposedThus, the empirical formulae for the concrete subjected to projectile impact predicts the penetration depth according to the projectile nose shape, and the scabbing depth is predicted by the predicted value of the penetration depth. Prior studies mainly investigated the improvement of the prediction accuracy concerning normal concrete without fiber reinforcement according to the projectile nose shape and analyzed the fracture properties of concrete, which were not present in the empirical formula, using the projectile nose shape.However, the behavior of the fracture on the rear face of the concrete caused by the projectile impact is affected by the stress wave transmitted to the rear face of the concrete. Therefore, the generation and transfer distribution of the impact stress wave differ depending on the projectile nose shape and considerably influence the fracture on the rear face of the concrete.In addition, fiber-reinforced concrete restrains the fracture on the rear face of the concrete by the offset of the impact stress wave and the reduction in the tensile strain caused by the improvement of the flexural tensile performance and toughness (energy-absorbing capacity) ,19.In this study, the fracture pattern and the crack distribution on the cross section of the concrete were observed after impact by three types of projectile  on nonreinforced and hooked steel fiber-reinforced concrete. The fracture depth and the crater diameter were evaluated. Furthermore, an analysis of the strain behavior on the rear face of the concrete and the distribution of the impact stress according to the projectile nose shape was performed.3 and a water absorption of 0.81% was used as the fine aggregate. Crushed gravel with a density of 2.65 g/cm3, a water absorption of 0.76%, and a maximum size of 20 mm was used as the coarse aggregate. A polycarboxylic acid-based superplasticizer was used to achieve sufficient concrete slump. The hooked steel fiber had a length of 30 mm, a diameter of 0.5 mm, an aspect ratio of 60, and a tensile strength of 1140 MPa. A twin-shaft mixer was used for concrete mixing. After dry mixing for 30 s with aggregate and binder, the process was finished by dry mixing for 90 s with the hooked steel fibers. Final mixing was then carried out for 300 s with admixture and water. Concrete compaction was performed by a vibrator. A slump value of approximately 150 mm was secured in all fresh concrete. Segregation was not observed.D1 and D2, respectively).S0) on the front face of the concrete panel to check the initial impact time. Seven additional gauges (S1–S7) were attached to the rear face of the concrete panel at distances of 0–285 mm from the center of the rear face. The sampling rate for the strain measurements was set to 200,000 Hz.The sharp nose shape caused the deepest penetration because of its sharp tip and small surface area. Conversely, the flat nose shape caused the shallowest penetration. The penetration depth for the hemispherical nose shape was between the sharp and flat penetration depths. Each projectile nose shape produced a similarly shaped indentation in the concrete.For the 60 mm thick specimens, scabbing occurred in the NC panels impacted by the hemispherical and flat projectile impacts. Scabbing did not occur following the sharp projectile impact. The impact by the hemispherical nose shape resulted in a scabbing area larger than that caused by the flat nose shape. Scabbing did not occur in the HSFRC panels because of the higher flexural tensile strength ,28,29.The cracks inside the HSFRC, which were caused by the projectile impact, were reduced because the flexural tensile performance was improved by the reinforcing fibers. In the case of the hemispherical nose projectile, scabbing was restrained because the large slope crack could not reach the backside.The measured penetration depths for the various projectile nose shapes were compared with the values predicted using the modified NDRC and UKAEA formulae . As presThe hemispherical projectile nose shape generated the largest rear-face crater diameter. Given its large impact area and shallower penetration, the rear-face crater diameter generated by the flat nose shape was thought to be smaller because the impact force transmitted to the rear face was distributed over a larger area. Conversely, the rear-face crater diameter generated by the sharp nose shape was thought to be the smallest because the impact force was concentrated in a smaller area, given its small impact area and deep penetration.Irrespective of the projectile nose shape, the front- and rear-face crater diameters were smaller for the HSFRC panels than for the NC panels. This result was attributed to the higher flexural tensile strength and the lower potential for scabbing in the fiber-reinforced concrete.S1 gauge (0 mm from the specimen center) was broken by concrete cracking. Cracking occurred inside the specimen near the S2–S3 gauges (50–95 mm from the specimen center), and the tensile strain was minimal near the S5–S7 gauges (190–285 mm from the specimen center). Similar to that of NC, the tensile strain of the S1 gauge for the HSFRC was high because of the occurrence of cracks from the concentrated impact stress at the center on the rear face of the concrete. However, the tensile strain sharply decreased after the S1 gauge.S1–S3 gauges (0–95 mm from the specimen center) were broken by the large tensile strain, and the scabbing area was the largest. Compared to that of NC, the tensile strain for the HSFRC was observed in a narrow area because the impact stress affecting the center on the rear face of the concrete was reduced by the fiber reinforcement.Following the hemispherical projectile impact, the S3 and S4  was lower than the strains owing to the sharp and hemispherical projectile impacts, but higher for S5–S7 (190–285 mm from the specimen center). The peak tensile strain caused by the flat projectile impact in S5–S7 was relatively higher than that of the sharp and hemispherical projectile impacts because the impact stress was dispersed in a wide range owing to the large impact area of the flat projectile. For the HSFRC, a tensile strain relatively higher than those of the sharp and hemispherical nose projectiles occurred at the S3 gauge.The tensile strain caused by the flat projectile impact at The impact force for the sharp nose shape was concentrated at the middle of the rear face, but scabbing did not occur because the concentrated area of the impact stress was lower in the 60 mm thick specimens compared to that in the 50 mm thick specimens. The impact force for the hemispherical nose shape was also concentrated at the middle, but over a larger area than in the sharp impact case. Meanwhile, the impact stress for the flat nose shape was distributed to the edges of the specimens.The strain behavior and the stress wave transmitted to the rear face varied based on the projectile nose shape and were thought to have a significant effect on the crack distribution of the cross section of the concrete panel and the crater diameter on the rear face of the concrete. Irrespective of the projectile nose shape, the overall tensile strain magnitude was lower for the HSFRC than for the NC panel. The reinforcing fibers were thought to absorb the impact force, thereby reducing the overall tensile strain.The sharp projectile nose resulted in a deeper penetration than hemispherical and flat projectile nose by up to about 36% because of the concentration of the impact force. Conversely, the flat projectile nose resulted in shallower penetrations because of the distribution of the impact force. In addition, the scabbing depth and crater diameter on the rear face of the concrete were larger for hemispherical projectile impact than for other projectiles. Therefore, if the scabbing occurs in concrete due to projectile impact, it is expected that the amount of the fragment by hemispherical projectiles will be greatest.The tensile strain on the rear face obtained from the experimental result was widely distributed as the projectile nose shape became blunter. The strain history on the rear face also differed in each projectile impact because the propagation path of the impact stress that transmitted to the rear face of the concrete was changed by the projectile nose shape. Therefore, the projectile nose shape had a significant effect on the crack distribution and the crater diameter on the rear face of the concrete.The penetration based on different projectile nose shapes was directly related to the impact force transmitted to the rear face. Considering the additional effect of the tensile strain on the rear face in predicting the fracture behavior, the scabbing caused by the projectile nose shape can more accurately be predicted. Furthermore, the crater diameter on the rear face of the concrete can be predicted.The tensile strain on the rear face of the concrete was reduced by the reinforcement of the hooked steel fiber because of the absorption of the impact stress transmitted to the rear face of the concrete by the hooked steel fiber. Furthermore, the strain history on the rear face is thought to more effectively reflect the deformability of the fiber-reinforced concrete owing to the impact load.This study evaluated the influence of projectile nose shape on the strain behavior and fracture properties of fiber-reinforced concrete panels due to projectile impact. The following conclusions can be drawn:


IgG4-related disease (IgG4-RD) is a fibroinflammatory condition. T-cells play a crucial role in the pathogenesis, and therefore, serum soluble interleukin-2 receptor (sIL-2R) may be a potential biomarker. n = 78) and controls (n = 101) and the serum sIL-2R levels in patients after treatment of IgG4-RD (n = 15). The disease activity was measured using the IgG4-Related Disease Responder Index (IgG4-RD RI).  We studied the levels of sIL-2R in 26 histologically proven IgG4-RD patients with available serum sIL-2R and compared them to those in newly diagnosed and untreated sarcoidosis patients (P < 0.001) and 6050 pg/ml in sarcoidosis patients (P = 0.004 compared to IgG4-RD). All IgG4-RD patients had elevated serum sIL-2R levels compared to the reference value of <2500 pg/ml in controls and 85% elevated serum IgG4; however, these did not correlate with each other. Both serum sIL-2R and IgG4 levels declined significantly after treatment . Before treatment, serum sIL-2R level and IgG4-RD RI did not correlate with each other. However, the decrease in serum sIL-2R upon treatment did correlate significantly (P = 0.04) with the decrease in disease activity assessed by IgG-RD RI.  Median serum sIL-2R in IgG4-RD patients was 4667 pg/ml compared to 1515 pg/ml in controls ( Serum sIL-2R is elevated in IgG4-RD reflecting the inflammatory process with enhanced T-cell activation. Furthermore, serum sIL-2R might serve as a potential marker of response to treatment in IgG4-RD. IgG4-related disease (IgG4-RD) is a systemic fibroinflammatory condition characterized by storiform fibrotic lesions and accumulation of IgG4-producing plasma cells in the affected tissues –3. It maβ, derived from T-helper 2 (Th2) cells and regulatory T-cells, may also contribute to the pathophysiology of IgG4-RD [The pathogenesis of IgG4-RD is mostly unclear, but B cells, IgG4-positive plasma cells, and IgG4 antibodies as well as the oligoclonal expansion of T-cells seem to play an important role in the immunopathophysiology –9. T fol IgG4-RD –13. Howe IgG4-RD , 15. Als IgG4-RD , 16.Peripheral blood levels of soluble IL-2 receptor (sIL-2R) reflect the level of T-cell activation. Elevated serum levels of sIL-2R correlate with disease activity in rheumatoid arthritis and sarcoidosis, diseases in which enhanced T-cell activity is centrally involved –20. MoreHere we examined sIL-2R serum levels in a cohort of histologically diagnosed and therapy-naïve patients with IgG4-RD in comparison to patients with untreated sarcoidosis and controls (blood bank donors). Moreover, serum sIL-2R levels were related to clinical response upon treatment.n = 78, from the Erasmus MC University Medical Center sarcoidosis database) and controls . Serum sIL-2R levels in untreated IgG4-RD patients were compared to serum sIL-2R levels after starting treatment (available in 15 patients). The disease activity at the time of measurement of serum sIL-2R levels before and after starting treatment was assessed using the IgG4-RD Responder Index (IgG4-RD RI) [The Erasmus MC University Medical Center represents a national referral center for patients with IgG4-RD. Medical records of patients with IgG4-RD between 1999 and July 2017 were reviewed for clinical characteristics and the availability of serum sIL-2R measurements. Only patients with histologically confirmed IgG4-RD according to established Boston criteria for histology , and of 4-RD RI) . This stSerum IgG4 and sIL-2R levels were measured by the standard laboratory diagnostic facility within Erasmus MC. IgG4 was measured by immunonephelometry using a Siemens BN II nephelometer, and serum sIL-2R levels were determined with ELISA  according to manufacturer instructions in pg/ml. The reference range of sIL-2R is set at <2500 pg/ml within Erasmus MC, based on the serum sIL-2R measurements in 101 blood bank donors. C-reactive protein (CRP) was determined from plasma on a Roche Cobas 8000 analyzer using an immunoturbidimetric assay (Roche Diagnostics). In short, human CRP agglutinates with latex particles coated with monoclonal anti-CRP antibodies. The aggregates are determined turbidimetrically.t-tests. Because we used multiple t-tests, this could be classified as multiple testing. Therefore, we used a stricter P value for those 3 specific tests (P < 0.01 is considered significant). For all other statistical tests performed, we used the standard P value (P < 0.05 is considered significant). To investigate whether serum sIL-2R levels changed after treatment, a dependent sample t-test was performed. When calculating correlations, we used the Spearman's rank correlation coefficient. The statistical analyses were performed using IBM SPSS statistics 21.0.0 for Windows .Characteristics of the patients with IgG4-RD and sarcoidosis and the controls were described using descriptive statistics. We tested for differences between the three groups of IgG4-RD, sarcoidosis, and controls using a one-way ANOVA. To investigate the individual differences between the groups , we used independent sample P = 0.311). Significantly more patients with IgG4-RD were male (76.9%). The blood bank donors were anonymous. Therefore, no data was present on the age or sex of these volunteers.P < 0.001) and lower compared to those in sarcoidosis (P = 0.004)  .P = 0.001). The duration of treatment varied per patient, but all patients of whom serum sIL-2R levels were available after treatment showed significant clinical improvement on IgG-RD RI in response to treatment  to 2864 pg/ml (2160–3653) (P = 0.001) from 4.0 g/l (1.7–10.3) to 1.8 g/l (1.3–3.3) and correlated with a clinical improvement of the disease. In all IgG4-RD patients, serum sIL-2R was elevated, compared to 85% (22/26) for serum IgG4 (P = 0.776) and after  treatment. Elevated CRP levels (CRP ≥ 10 mg/l) were found in 27% (7/26) of patients before treatment. No significant correlation was observed between the levels of CRP and serum sIL-2R before  and after (Spearman's rho: 0.379 P = 0.164) treatment. Serum sIL-2R and IgG4-RD RI did not correlate with each other before treatment . However, the decrease in serum sIL-2R significantly correlated with the decrease in disease activity assessed by IgG-RD RI (P = 0.044).In 19 patients with IgG4-RD, serum IgG4 levels were available before and after treatment and decreased significantly  and elderly (age 67–99 years) having higher sIL-2R levels as compared to (young) adults age 22–67) , 25. We –67 , 25.Our observations in patients with clinically active IgG4-RD suggest that serum sIL-2R reflects the inflammatory process in these patients. sIL-2R is secreted by activated T-cells, and elevated levels are used as marker for T-cell activity in other inflammatory diseases . AlthougTheoretically, serum IgG4 levels might reflect disease activity in IgG4-RD. However, in general, serum IgG4 is normal in ~30% of IgG4-RD patients . Indeed Sarcoidosis often presents with clinical presentations similar to IgG4-RD and is associated with elevated serum sIL-2R (this study and others), and if untreated, fibrosis may develop , 18, 29.This study is limited by its retrospective character and the relatively small population. Therefore, larger studies are required to obtain the sensitivity and specificity of sIL-2R in IgG4-RD.In conclusion, we demonstrate that serum sIL-2R is elevated in IgG4-RD. Furthermore, serum sIL-2R may have potential as a tool for monitoring disease activity/treatment response in IgG4-RD. The value of serum sIL-2R for this application needs further confirmation in prospective and larger studies, also in comparison to diseases with the capacity to mimic IgG4-RD such as granulomatosis with polyangiitis.