Monascus pigments are widely used in the food and pharmaceutical industries due to their safety to human health. Our previous study found that glucose concentration induced extracellular oxidoreduction potential (ORP) changes could influence extracellular water-soluble yellow pigment production by Monascus ruber CGMCC 10910 in submerged fermentation. In this study, H2O2 and dithiothreitol (DTT) were used to change the oxidoreduction potential for investigating the effects of oxidative or reductive substances on Monascus yellow pigment production by Monascus ruber CGMCC 10910.2O2 and DTT. Both cell growth and extracellular water-soluble yellow pigment production were enhanced under H2O2-induced oxidative (HIO) conditions and were inhibited under dithiothreitol-induced reductive conditions. By optimizing the amount of H2O2 added and the timing of the addition, the yield of extracellular water-soluble yellow pigments significantly increased and reached a maximum of 209 AU, when 10 mM H2O2 was added on the 3rd day of fermentation with M. ruber CGMCC 10910. Under HIO conditions, the ratio of NADH/NAD+ was much lower than that in the control group, and the expression levels of relative pigment biosynthesis genes were up-regulated; moreover, the activity of glucose-6-phosphate dehydrogenase (G6PDH) was increased while 6-phosphofructokinase (PFK) activity was inhibited.The extracellular ORP could be controlled by H2O2 increased water-soluble yellow pigment accumulation via up-regulation of the expression levels of relative genes and by increasing the precursors of pigment biosynthesis through redirection of metabolic flux. In contrast, reductive conditions induced by dithiothreitol inhibited yellow pigment accumulation. This experiment provides a potential strategy for improving the production of Monascus yellow pigments.Oxidative conditions induced by HThe online version of this article (10.1186/s12934-017-0828-0) contains supplementary material, which is available to authorized users. Monascus pigments are mixed pigments with three main colours: yellow, orange and red [Monascus yellow pigments have been widely researched due to their anti-tumour [Monascus yellow pigments are beneficial to human health in appropriate doses and can potentially be used as nutraceutical or therapeutic agents. and red . Recentli-tumour , 3, antii-tumour , anti-obi-tumour , anti-dii-tumour , and thei-tumour , counteri-tumour , and trei-tumour . A largeMonascus yellow pigments generally consist of a large amount of intracellular alcohol-soluble pigments and a small amount of extracellular water-soluble pigments [Monascus anka [M. ruber CGMCC 10910 using high glucose concentrations with low oxidoreduction potential (ORP) [In submerged fermentation, pigments . Water-spigments . Water-spigments , but thipigments , 14, strpigments , gene mopigments , and medpigments , 18. Checus anka . In addial (ORP) , 21. How2O2, dithiothreitol (DTT), and potassium ferricyanide [Saccharopolyspora spinosa fermentation under HIO conditions, and high citric acid productivity could be achieved in Aspergillus niger cultures with defined ORP profiles [Monascus pigment biosynthesis occurs via a polyketide pathway and requires many primary metabolites, such as acetyl-CoA, malonyl-CoA, and NADPH [Monascus pigments in the M. purpureus and M. ruber genome and the functions of some critical genes involved in the pigment biosynthetic pathway were reported [MpPKS5 (the homolog of MpigA in M. ruber) in M. purpureus and the targeted-deletion of MpigA in M. ruber resulted in the abolishment of pigment production, confirming that polyketide synthase is involved in pigment biosynthesis [MpFasA2 (the homolog of MpigJ in M. ruber) and MpfasB2 (the homolog of MpigK in M. ruber) respectively encode fatty acid synthase α-subunit and fatty acid synthase β-subunit and supply the medium-chain (C8 and C10) fatty acyl moieties for Monascus pigments biosynthetic [mppA (the homolog of MpigC in M. ruber), mppC (the homolog of MpigE in M. ruber) and mppE (the homolog of MpigG in M. ruber) mutants of M. purpureus substantiate that MppA-mediated ω-2 ketoreduction is a prerequisite for the synthesis of the pyranoquinone bicyclic core of the Monascus pigments and MppC activity determines the regioselectivity of the spontaneous Knoevenagel condensation. The mppB (the homolog of MpigD in M. ruber) in M. purpureus gene encodes a trichothecene 3-O-acetyltransferase (AT), which can transfer the medium-chain (C8 and C10) fatty acyl group into the polyketide chromophore to complete pigment biosynthesis. The mppD (the homolog of MpigF in M. ruber) gene in M. purpureus encodes an amine oxidase/esterase. The mppR1 (the homolog of MpigB in M. ruber) and mppR2 (the homolog of MpigI in M. ruber) genes in M. purpureus are regulatory genes encode transcription factors for pigments biosynthesis [Oxidoreduction potential was identified as a control parameter for fermentation processes –24. Intricyanide . It has profiles , 28. Thend NADPH , 31, 32.nd NADPH . Recentlreported , 31. Botynthetic . The proynthesis –36. Thos2O2 was added to the fermentation medium of M. ruber CGMCC 10910. Its concentration and addition time were also optimized to improve extracellular water-soluble yellow pigment production. In addition, the effect of the oxidative environment induced by H2O2 on the extracellular and intracellular ORP, the expression levels of pigment biosynthesis genes, and the activities of key enzymes (PFK and G6PDH) in the glycolysis and pentose phosphate pathway (PPP) were also investigated.In this study, HMonascus ruber CGMCC 10910, which was deposited at the China General Microbiological Culture Collection Center (CGMCC), was used in this study, and it was cultivated on a potato dextrose agar (PDA) medium at 30 °C for 7 days and then stored at 4 °C.2PO4, 0.05% KCl, and 0.001% FeSO4·7H2O. The fermentation culture medium contained 15% glucose, 0.5% (NH4)2SO4, 0.5% KH2PO4, 0.05% MgSO4·7H2O, 0.05% KCl, 0.003% MnSO4·H2O, 0.001% ZnSO4·7H2O, and 0.001% FeSO4·7H2O. For the seed culture, 5–6 loopfuls of single colonies (approximately 10 mm diameter) were scraped off an agar plate and inoculated into a 250 mL Erlenmeyer flask containing 50 mL of seed culture medium and agitated at 180 rpm at 30 °C. After 25 h, 2 mL of seed culture was used to inoculate a 250 mL Erlenmeyer flask containing 25 mL of fermentation culture medium, which was then fermented for 12 days with an agitation speed of 180 rpm at 30 °C. Three duplicates were carried out for each condition.The seed culture medium contained 2% glucose, 0.3% yeast extract, 1% peptone, 0.4% KH2O2 and DTT were filter-sterilized before being added to the medium. One, 5, 10, 15 or 20 mM H2O2 was added to the medium on day 2 of fermentation to study the effect of H2O2 concentration on cell growth and yellow pigment production. Ten millimolar H2O2 was added to the medium on day 0, 1, 3, and 5 of fermentation to study the effect of H2O2 addition time on cell growth and yellow pigment production. Three gram per liter DTT was added to the medium on day 3 to study the extracellular reducing conditions on cell growth and yellow pigment production.HAfter being cultured, the fermentation broth was vacuum-filtered through a 0.8 mm mixed cellulose esters membrane. The filtrate (extracellular broth) was appropriately diluted to determine the residual glucose concentration using the standard 3,5-dinitrosalicylic acid (DNS) method. Extracellular ORP was detected via an oxidation–reduction electrode  . After fThe filtrate (extracellular broth) was appropriately diluted to determine the extracellular water-soluble yellow pigment concentration. The absorbance spectrum of the water-soluble yellow pigments was recorded by a UV–visible spectrophotometer  from 300 to 550 nm at 1 nm intervals, and the absorbance units (AU) at the peak wavelength of 350 nm was multiplied by the dilution ratio, which was used as an index for the water-soluble yellow pigment concentration .The intracellular pigment concentration was determined according to the following the procedure: the harvested and washed mycelia were resuspended in 25 mL acidic aqueous ethanol  and incubated for 1 h. The suspension was filtered with filter paper, and the filtrate (intracellular extract) was appropriately diluted to determine intracellular pigment concentration. The absorbance spectrum of the intracellular pigments was recorded by a UV–visible spectrophotometer  from 300 to 550 nm at 1 nm intervals, and the AU at the peak wavelength of 410 nm were multiplied by the dilution ratio, which was used as an index for the intracellular yellow pigment concentration.3PO4 solution  and acetonitrile (phase B) were used as the mobile phase using the following gradient program: 0 min, 80% A, 20% B; 25 min, 20% A, 80% B; 35 min, 20% A, 80% B; 36 min, 80% A, 20% B; 41 min, 80% A, 20% B. The PDA was set at 200–600 nm, and the flow rate of the mobile phase was 0.8 mL/min [Analyses of pigment compositions were performed using an Alliance e2695 HPLC system  equipped with a 2998 Photodiode Array (PDA) detector  and a Zorbax Eclipse Plus C18 column . The temperature of the column oven was set at 30 °C. A mixture of H8 mL/min .LC–MS (liquid chromatography–mass spectrometry) consisted of a HP1100 HPLC system  and a microTOF-QII mass spectrometer . The C18 column and chromatographic conditions were the same as mentioned above, except for mobile phase A .g, 4 °C for 10 min. The supernatant transformed into a tube and was neutralized using alkaline extracting solution or acidic extracting solution. After neutralization, the samples were centrifuged at 12,000g, 4 °C for 10 min. The supernatant was collected for NAD+ and NADH determination using the Coenzyme I NAD(H) content test kit  according to the manufacturer’s instructions. The kit is based on an enzymatic cycling assay method.The intracellular NADH and NAD+ concentrations were determined using procedures described in previous studies , 37. Myc2, 20 mM NH4Cl, 0.5 mM DTT) and then fully shocked [Mycelia were rapidly ground into a powder using liquid nitrogen; 0.1 g of mycelia powder was quickly transferred into a sample tube containing 1 mL of crude enzyme extracting solution (25 mM Tris–HCl (pH = 7.6), 10 mM MgCl shocked , 39. TheMpFasA2, MpFasB2, MpPKS5, mppR1, mppA, mppB, mppC, mppD, mppE, mppR2 (GenBank Accession No. KC148521) and the actin gene (GenBank Accession No. AJ417880) are listed in Additional file The relative expression levels of key genes involved in pigment biosynthesis were analysed using real-time quantitative PCR as described previously . Myceliap < 0.05 and p < 0.01 were considered significant and highly significant, respectively.Each experiment was repeated in triplicate, at minimum. Numerical data are presented as the mean ± SD. The differences among the various treatments were analysed using one-way ANOVA. All statistical analyses were performed by using SPSS 22.0 software, and Monascus ruber CGMCC 10910 has the potential to produce extracellular water-soluble yellow pigments with a maximum absorbance wavelength of 350 nm [350 under high glucose stress fermentation [2O2 to the medium on the 2nd day of M. ruber CGMCC 10910 fermentation under high glucose stress are shown in Fig. 2O2 could promote cell growth while higher amounts of H2O2 had an inhibitory effect on cell growth , and a 35 and 26% increase in extracellular and intracellular yellow pigment yield, respectively. Thus, 10 mM was chosen as the suitable concentration for the addition time experiments.f 350 nm , and theentation . Further2O2, was added to the fermentation medium at various growth time points, including at day 0 (beginning of fermentation), the 1st day , the 3rd day , and the 5th day  during pigment fermentation. Cell growth and pigment production, including intracellular and extracellular yellow pigments, were inhibited when H2O2 was added at the beginning of fermentation , in which extracellular water-soluble yellow pigment yield reached approximately 209 AU350, 42% higher than that of the control , while dithiothreitol (DTT) acting as a reducing agent could reduce the extracellular ORP [2O2 was added to the medium on the 3rd day to create an extracellular oxidative environment, and 3 g/L DTT was added on the 3rd day to create an extracellular reducing environment. Extracellular ORP increased immediately after H2O2 addition and reached its highest level on day 4, then decreased to a low level, similar to the control, in the later phase of the fermentation  fatty acyl moieties, and then a trichothecene 3-O-acetyltransferase (AT), encoded by the mppB gene, transfers the medium-chain (C8 and C10) fatty acyl groups onto the polyketide chromophore to complete pigment biosynthesis. mppA, mppC, and mppE encode oxidoreductases, of which a reductive enzyme encoded by mppE controls the biosynthesis of yellow pigments. The gene mppD encodes an amine oxidase/esterase. The genes mppR1 and mppR2 are regulatory genes for pigment biosynthesis [The pigment biosynthetic gene cluster has been identified in cus spp. . The expynthesis , 32.Fig.MpFasA2, MpFasB2, MpPKS5, mppA, mppB, mppD, mppE, and mppR1 were significantly up-regulated (p < 0.01 or p < 0.05) under HIO conditions  and were negatively correlated with pigment production. It has been reported that mppR2 is a negative regulatory factor [The expression levels of the genes y factor .M. ruber CGMCC 10910 caused by HIO conditions, the activities of key redox-dependent enzymes (PFK and G6PDH) in glycolysis and the pentose phosphate pathway (PPP) were analysed. As shown in Fig. 2O2. At the same time, G6PDH activity was enhanced between the 4th day and the 8th day under HIO conditions, which also indicated that the PPP was enhanced under HIO conditions  [2O2 was added at different fermentation time points to study the effects of H2O2 addition time on cell growth and yellow pigment production in M. ruber CGMCC 10910. Cell growth and yellow pigment production were both inhibited when H2O2 was added at the beginning of fermentation  , 21. In M. ruber . Cell grcus 5008 , 45. Bec2O2 is an electron acceptor, which can induce an extracellular oxidative environment (higher extracellular ORP) when added to the fermentation medium [2O2 and DTT were used to modify the extracellular ORP to study the effect of extracellular ORP changes on the water-soluble yellow pigment production in M. ruber CGMCC 10910. The results showed that the yield of yellow pigments (both extracellular and intracellular) and cell growth under oxidative conditions were significantly increased , were not needed to balance NADH/NAD+ metabolism under HIO conditions [2O2 regulating NADH/NAD+ ratios maybe one of the reasons why DCW and yellow pigment yield were increased under HIO conditions  could be produced for polyketide and lipid biosynthesis [Streptomyces hygroscopicus 5008 fermentation in response to various oxidant treatments [MpFasA2, MpFasB2, MpPKS5, mppA, mppB, mppD, mppE, and mppR1 were significantly up-regulated under HIO conditions (p < 0.01 or p < 0.05)  was decreased by H2O2 addition; fewer metabolites were used to balance NADH/NAD+, and more energy could thus be directed to cell growth and yellow pigment production instead of unusable metabolite production. The transcription levels of relevant pigment biosynthesis genes were up-regulated under HIO conditions; moreover, the activity of G6PDH was increased while PFK activity was inhibited. HIO conditions increased yellow pigment accumulation via up-regulation of the transcription levels of relevant genes and improving the precursor concentrations of yellow pigment biosynthesis through redirection of metabolic flux, which provides a potential improved strategy for producing water-soluble Monascus yellow pigments.Extracellular oxidative conditions caused by HAdditional file 1: Table S1. Primers for RT-qPCR analyzing pigments biosynthetic genes.Additional file 2: Figure S1. Mass spectra and UV–visible spectra of extracellular yellow pigments detected by LC–MS and HPLC-PDA.Additional file 3: Figure S2. Mass spectra and UV–visible spectra of intracellular yellow pigment monascin and ankaflavin detected by LC–MS and HPLC-PDA.Additional file 4: Figure S3. HPLC-PDA chromatogram of intracellular yellow pigments and monascin standard curve detected by HPLC-PDA.


Arabidopsis thaliana via changes in splicing of key regulatory genes. Here we investigate whether a particular histone modification affects ambient temperature-induced alternative splicing and flowering time.Global warming severely affects flowering time and reproductive success of plants. Alternative splicing of pre-messenger RNA (mRNA) is an important mechanism underlying ambient temperature-controlled responses in plants, yet its regulation is poorly understood. An increase in temperature promotes changes in plant morphology as well as the transition from the vegetative to the reproductive phase in We use a genome-wide approach and perform RNA-sequencing (RNA-seq) analyses and histone H3 lysine 36 tri-methylation (H3K36me3) chromatin immunoprecipitation sequencing (ChIP-seq) in plants exposed to different ambient temperatures. Analysis and comparison of these datasets reveal that temperature-induced differentially spliced genes are enriched in H3K36me3. Moreover, we find that reduction of H3K36me3 deposition causes alteration in temperature-induced alternative splicing. We also show that plants with mutations in H3K36me3 writers, eraser, or readers have altered high ambient temperature-induced flowering.Our results show a key role for the histone mark H3K36me3 in splicing regulation and plant plasticity to fluctuating ambient temperature. Our findings open new perspectives for the breeding of crops that can better cope with environmental changes due to climate change.The online version of this article (doi:10.1186/s13059-017-1235-x) contains supplementary material, which is available to authorized users. Plants sense changes in the environment and adapt their development accordingly. Temperature is one of the environmental signals that strongly affects plant development. For example, plants are able to adapt their organ shape in relation to the temperature they experience, a phenomenon called “thermo-morphogenesis” . As suchA change in ambient temperature affects gene functioning at transcription level and at post-transcriptional level, e.g. through protein conformation and stability. Recently, it has been shown that ambient temperature affects phytochrome B (PHYB) inactivation during the night . MoreoveArabidopsis thaliana.In plants, it is not known if a similar link between chromatin and splicing exists and whether such a mechanism would be ambient temperature sensitive. H3K36me3 was found to be prevalent in transcribed regions and therefore a function in transcriptional elongation has been postulated for this histone mark . Here weTo study the proposed linkage between the H3K36me3 mark and ambient temperature-induced AS, we first identified transcriptome-wide differentially spliced (DiS) events upon a temperature change and investigated the temporal dynamics of this process. Arabidopsis plants were grown at 16 °C in short day conditions (8 h light-16 h dark) for five weeks and then transferred to 25 °C. With RNA sequencing (RNA-seq), we analyzed the changes in the transcriptome at one, three, and five days after the temperature change. This experimental setup allows to monitor initial changes in splicing pattern upon a temperature change and to investigate the stability of these effects. For each time point, we quantified AS setting one isoform as reference and then calculating the “Percentage of Spliced In” (PSI) for each type of event  [MADS AFFECTING FLOWERING 2 (MAF2) [PSEUDO-RESPONSE REGULATOR 3 (PRR3) and PRR7 [FLM, we observed an increase in the retention of the second intron and a MXE event [MAF2 locus generates an isoform with a premature stop codon that is most likely targeted for NMD and the increased abundance of this isoform at higher temperature leads to a decrease of the functional flowering time repressor [PRR3 and PRR7 were shown previously to undergo AS upon a change to low temperature from 20 °C to 4 °C [PRR7 as previously reported for a change to low temperature, as well as new events for PRR7 and PRR3 that appear to be specific to the increase in ambient temperature in our experimental setup  , 21 and 2 (MAF2) , 34 , SERINE/ARGININE-RICH PROTEIN SPLICING FACTOR 34 (SR34), and the U2 snRNP auxilliary factors U2AF65A and U2AF65B.Moreover, we found splicing regulators DiS upon the temperature change, such as S40 Fig. , SC35-LISQUAMOSA PROMOTER BINDING PROTEIN-LIKE 2 (SPL2) [MODIFIER OF SNC 1 (MOS1) [AUXIN RESPONSE FACTOR 2 (ARF2) [PIN-FORMED 7 (PIN7) [VIN3-LIKE 2 (VIL2) [SPLAYED (SYD) [Additionally, we also identify novel temperature-induced splicing events in master regulators of various biological processes, such as the flowering time regulator 2 (SPL2)  and MODI1 (MOS1) , the reg2 (ARF2)  and PIN-7 (PIN7) , and the2 (VIL2)  and SPLAED (SYD) –43   Fig. .Fig. 2TePIN7 and VIL2 at 25 °C compared with 16 °C  loci  and H3K36me3-independent if the events are not affected by the loss of H3K36me3 . H3K36me3-dependent events tend to be located closer to an H3K36me3 region compared with H3K36me3-independent events  at both temperatures  [jmj30-1 is more sensitive to the temperature while JMJ30-OE is less responsive in temperature-induced flowering    and MRG2 are able to bind to H3K36m3 modified histones, as is the case for the human MRG15 [PTB homologs in Arabidopsis , reveals their role in the regulation of splicing of flowering time genes, including FLM [MRG1 and MRG2 proteins play a pivotal role. In agreement with this model, we found that mrg1-1 mrg2-3 mutant plants are less sensitive to temperature-induced flowering than the WT . Nevertheless, the preferential location of the H3K36me3 mark in the proximity of H3K36me3 dependent AS events could also point to an effect on transcription speed in support of the kinetic model. In agreement, transcription rate was found to be affected by temperature in Arabidopsis [While our findings highlight a key role for H3K36me3 in the regulation of ambient temperature-induced splicing, the exact molecular mode of action still remains to be elucidated. Our results do not exclude the kinetic model, neither the adapter-complex model, which have been proposed to explain the linkage between chromatin modifications and AS , 31. Insbidopsis . Additiobidopsis , was recbidopsis , 60. TheVIL2 regulates the ambient temperature-associated flowering time gene MAF5 [We show that H3K36me3 plays a key role in the regulation of temperature-induced flowering. Flowering time is a key developmental process, because a proper timing in the switch from the vegetative to the reproductive phase ensures plant fitness. We found that key regulators in the ambient temperature-controlled flowering time pathway are subject to H3K36me3 level dependent AS. However, H3K36me3 may also affect temperature-induced flowering indirectly by affecting splicing of upstream regulators of flowering time genes. For example, the temperature DiS gene ene MAF5 . AlternaAS of pre-mRNA is a key mechanism to generate protein diversity or to regulate transcript isoform abundance and it allows rapid changes in the transcriptome. In temperature sensing, AS represents a fast and flexible way to confer plasticity in plant development. The co-transcriptional nature of splicing creates opportunities for cross-regulation between splicing and chromatin landscape. In mammals, histone modifications contribute to the regulation of splicing. Here we show a direct role for histone modifications in AS regulation in plants. We reveal a key role for H3K36me3 in the control of temperature-induced AS and in temperature-dependent flowering time control in Arabidopsis.Besides that AS of key regulatory genes confers plasticity, because it is a fast and reversible response to changing environmental conditions, our findings on the role of H3K36me3 in this process open interesting prospective for a memory of temperature mediated AS. It is tempting to speculate that temperature variation is memorized in the chromatin landscape via H3K36me3 deposition, leading to a specific splicing pattern, which allows the memory of environmental conditions during the life cycle and a possible adaptation to a rapidly changing climate.sdg8-2 Salk_026442 [sdg26-1 Salk_013895 [jmj30-1 (N67787) [mrg1-1 mrg2-3 [Seeds of knock-out lines, k_026442 , sdg26-1(N67787) , and the(N67787) , were ob1 mrg2-3  were a gThree biological samples were generated for each condition. For each sample, tissue from approximately ten plants was collected 4 h after light on for the time-series experiment and between 4 h and 7 h after light on for the mutant experiment. Using jeweler’s forceps, leaves were removed to obtain SAM enriched tissue. Total RNA was extracted using the InviTrap Spin Plant RNA Mini Kit (REF: 1064100300) from Invitek according to the manufacturer’s protocol. DNAse treatment was performed to remove genomic DNA. DNase I digestion was performed on total RNA using Turbo DNase from Ambion according to the manufacturer’s protocol. RNA integrity was checked on 1% (w/v) agarose gel after DNase I treatment. Samples were prepared for Illumina sequencing using the TruSeq Stranded mRNA Sample Prep kit (REF: 15032613) from Illumina according to the manufacturer’s protocol. Libraries were analyzed on the Bioanalizer and quantified with the qBit before pooling for sequencing on HiSeq2500. Two lanes on a 125pb PE run were used.Arabidopsis thaliana genome version TAIR10 (www.arabidopsis.org) using TopHat2 [RNA-seq reads were mapped against the  TopHat2 . To impr TopHat2 . Cuffmer TopHat2  was usedTo retrieve differentially expressed genes upon the temperature changes, the number of fragments mapping to TAIR10 annotated genes was determined using HTseq count . Differep value < 0.05 and at least five genes were present.GO analysis was performed using the Cytoscape plugin BiNGO  using thFour independent biological replicates for each temperature were generated. For each sample, 0.5 g of plant material was used per biological replicate. Material was collected from plants growing at 16 °C, as well as from plants one day after the plants were moved to 25 °C (5–6 h after lights on). Using jeweler’s forceps, leaves were removed to obtain SAM-enriched tissue. ChIP experiments were performed following a previously published protocol . After thttp://deeptools.ie-freiburg.mpg.de/). Regions with very large counts were removed to avoid bias in the Pearson correlation  in short day conditions  in walk-in climate chambers with humidity of 65%. Trays were rearranged every two days and nutrient was supplied by sub-irrigation. After five weeks of growth at 16 °C, half of the plants were moved to the 25 °C growth chamber. Flowering time was quantified by determining the time until the macroscopic appearance of the first flower bud (DTB), screening was done every two days and by counting RLN after bolting of all plants in the tray. The experiment was performed in three biological replicates with 15 plants in each replicate for each genotype/condition. Data are shown as average of ratios between the replicates. Student’s t-tests were calculated with GraphPad QuickCalcs.Additional file 1: Table S1.List of significant see  DiS evenAdditional file 2: Supplementary Figure S1-S7.Figure S2. H3K36me3 dynamics upon the temperature change. Figure S3. Quantitative change in H3K36me3 (ChIP-seq output) at the same location of a DiS event (RNA-seq output) for the circadian clock genes PRR3 and PRR7. Figure S4. Temperature-induced AS in sdg8-2 and sdg26-1. Figure S5. Changes in H3K36me3 levels and mrg1 mrg2 affect temperature induction of flowering. Figure S6. Changes in H3K36me3 levels do not cause pleotropic effects on plant development. Figure S7. Pearson correlation of ChIP-seq experiments. (PDF 1186 kb)Temperature induced AS events. Additional file 3: Table S2.p value according to the BH method for controlling false discovery < 0.05. (XLSX 518 kb)DEGs between 16 °C and 25 °C day 1 (sheet1), 16 °C and 25 °C day 3 (sheet2), 16 °C and 25 °C day 5 (sheet3). DEGs are defined as genes with a change in expression of log2 Fold Change |1| and adjusted Additional file 4: Table S3.H3K36me3 modified regions at 16 °C or 25 °C day 1 and quantitative comparison obtained with DANPOS2. (XLSX 15623 kb)Additional file 5: Table S4.List of significant see “” DiS eveAdditional file 6: Table S5.sdg8-2 and events between WT and sdg26-1 at 16 °C. (XLSX 17 kb)List of significant see “” DiS eve


Thus, the hDRG provides a translational model in which to study “human pain in a dish” and test new pain therapeutics.Voltage-gated sodium channels (Navs) play an important role in human pain sensation. However, the expression and role of Nav subtypes in native human sensory neurons are unclear. To address this issue, we obtained human dorsal root ganglion (hDRG) tissues from healthy donors. PCR analysis of seven DRG-expressed Nav subtypes revealed that the hDRG has higher expression of Nav1.7  and lower expression of Nav1.8 (~12%), whereas the mouse DRG has higher expression of Nav1.8 (~45%) and lower expression of Nav1.7 (~18%). To mimic Nav regulation in chronic pain, we treated hDRG neurons in primary cultures with paclitaxel (0.1–1 μmol/L) for 24 h. Paclitaxel increased the Nav1.7 but not Nav1.8 expression and also increased the transient Na Despioroquine . Importaoroquine .SCN9A, the gene that codes for Nav1.7 in humans, result in a congenital inability to sense pain and anosmia without affecting other sensations such as touch and temperature [et al. showed that human Nav1.8 channels display slower inactivation kinetics and produce larger persistent current and ramp current than previously reported in other species [Voltage-gated sodium (Nav) channels play a critical role in the initiation and propagation of action potentials in excitable cells such as neurons and are the major targets of analgesics –14. Nineperature , 21. Conperature , 23. Des species . Our datPaclitaxel was purchased from Sigma . Specific primers were designed using the Real-Time PCR web tool from IDT SciTools .Adult CD1 mice  were obtained from Charles River Laboratories  and used for PCR analysis. All the animal procedures performed in this study were approved by the Animal Care Committee of Duke University Medical Centre.Human DRGs were obtained from healthy donors through the National Disease Research Interchange  with permission from the Duke University Institutional Review Board. Postmortem L3–L5 DRGs were dissected from donors  were cut on a cryostat. The sections were first blocked with 2% BSA for 1 h at room temperature, then incubated with peripherin  or a mixture of primary polyclonal TRPV1 antibody  and monoclonal NF200H antibody  overnight at 4 °C. The sections were then incubated for 2 h at room temperature with cyanine 3 (Cy3)- and FITC-conjugated secondary antibodies . The DRG sections were then stained with DAPI  and examined under a Nikon fluorescence microscope. Images were captured with a CCD Spot camera and analyzed with NIH Image J software or Adobe PhotoShop.−ΔΔCT method. The relative mRNA expression of each Nav subtype is shown as a percentage of the total Navs  tested in DRGs [DRG tissues were rapidly isolated in RNAse-free conditions. Total RNAs were extracted using an RNeasy Plus Mini kit . The quantity and quality of the eluted RNA samples were verified using a NanoDrop spectrophotometer . Total RNAs (0.5 μg) were reverse-transcribed using the QuantiTect Reverse Transcription Kit according to the protocol of the manufacturer (Qiagen). The sequences of human Nav primers are listed in Table  in DRGs .Table 2S2+-free Hank’s balanced salt solution (Thermo Fisher Scientific), and cultures were prepared as previously reported [2 incubator for 120 min with collagenase Type II  and dispase II  in phosphate-buffered saline with 10 mmol/L HEPES, pH adjusted to 7.4 with NaOH. hDRGs were mechanically dissociated using fire-polished pipettes, filtered through a 100-μm nylon mesh, and centrifuged for 5 min (500 g). The DRG cell pellet was re-suspended and plated on 0.5 mg/mL poly-D-lysine-coated glass coverslips. The DRG cultures were grown in Neurobasal medium supplemented with 10% fetal bovine serum, 2% B-27 supplement, and 1% penicillin/streptomycin.Upon delivery, DRGs were rapidly dissected from the nerve roots, minced in Careported . DRGs we+ currents and action potentials with an EPC10 amplifier  and an Axopatch-200B amplifier with a Digidata 1440A . The patch pipettes were pulled from borosilicate capillaries . The resistance was 3–4 MΩ when filled with pipette solution. The recording chamber (300 µL) was continuously superfused at 3–4 mL/min. Series resistance was compensated (>80%) and leak subtraction was performed. Data were low-pass-filtered at 2 kHz and sampled at 10 kHz. Patchmaster (HEKA) and pClamp10 (Axon Instruments) softwares were used during experiments and analysis. For recording Na+ currents, the pipette solution contained (in mmol/L): CsCl 130, NaCl 9, MgCl2 1, EGTA 10, HEPES 10, adjusted to pH 7.3 with CsOH. For action potential recordings, the pipette solution contained (in mmol/L): K-gluconate 136, NaCl 10, MgCl2 1, EGTA 10, HEPES 10, Mg-ATP 2, adjusted to pH 7.3 with KOH. According to our preliminary study in hDRG neurons, action potentials were induced by 500 pA current injection for 1 s. The external solution for action potential recordings was composed of (in mmol/L): NaCl 140, KCl 5, CaCl2 2, MgCl2 1, HEPES 10, glucose 10, adjusted to pH 7.3 with NaOH. The external solution for recording transient Na+ currents contained (in mmol/L): NaCl 131, TEACl 10, CsCl 10, CaCl2 1, MgCl2 2, CdCl2 0.3, 4-aminopyridine 3, HEPES 10, glucose 10, adjusted to pH 7.4 with NaOH. In voltage-clamp experiments, transient Na+ currents were evoked by a test pulse to 0 mV from the holding potential (−70 mV).Whole-cell patch-clamp recordings in small-diameter (<50 μm) hDRG neurons were conducted at room temperature. We used patch pipettes to record transient Napost-hoc Bonferroni test or unpaired two-tailed Student’s t-test. Pearson’s correlation was used to test the linear dependence of two variables. Statistical analyses were performed with Prism 6.0 (GraphPad Software) and the criterion for statistical significance was P < 0.05.All data are expressed as mean ± SEM. Biochemical and electrophysiological data were tested using one-way or two-way ANOVA followed by the n = 28–37 neurons/group). Our results are in agreement with previous reports that hDRG neurons with diameters <50 μm can be regarded as small nociceptive neurons [Immunofluorescent staining of hDRGs is a challenge, because of the high background of autofluorescence in human tissue . We were neurons .Fig. 1HiWe used quantitative real-time PCR (qPCR) to compare the transcriptional expression of seven Nav subtypes in hDRGs and mDRGs: TTX-sensitive Navs  and TTX-resistant Navs (Nav1.8 and Nav1.9). Notably, all these subtypes are known to be expressed in rodent DRG neurons , 16, 28.We also investigated the sex differences in Nav subtype expression in hDRG and mDRG tissues. The relative expression of each Nav subtype in the hDRGs of 6 men and 6 women showed no sex difference in the expression of any subtype Fig. A. SimilaPrevious studies have shown that Nav1.7 is upregulated in DRG neurons under chronic pain conditions such as diabetic neuropathy, and contributes to the pathogenesis of neuropathic pain , 29, 30.+ currents in small-diameter neurons (<50 μm). In untreated hDRG neurons, the amplitude of transient Na+ currents was 3131 ± 573.1 pA  in untreated hDRG neurons. After paclitaxel treatment , there was a slight decrease in rheobase . This lack of significance may result from the small number of neurons recorded. Current-clamp recording revealed that current injection  evoked action potentials in hDRG neurons  molecular mechanisms identified in rodents may not apply to humans and (2) animal models of pain may not represent human pains , 6. To fn autism .Nav1.7-knockout mice recapitulate the phenotype of human congenital indifference to pain [Although the role of human Nav1.7 has been well-studied in heterologous cells and rodent DRG neurons by over-expression of different human Nav1.7 mutants in these cells, the specific role of Nav1.7 in native hDRG neurons is unclear. Global  to pain . Nerve t to pain , 30. How to pain . Further to pain . Thus, tSCN9A [SCN10A (encoding Nav1.8) [SCN11A (encoding Nav1.9) [SCN8A (encoding Nav1.6) [+ current and excitability , in small-diameter (presumably nociceptive) hDRG neurons. Although other Nav subtypes could also contribute to the transient Na+ current, Nav1.7 is a major contributor to this TTX-sensitive current [Our data have shown striking species differences in the expression of different Nav subtypes. Especially, the ratio of Nav1.7 expression in the hDRG (50%) is much higher than in the mDRG (20%). On the other hand, the mDRG has a much higher ratio of Nav1.8 (45%) than the hDRG (15%), and a critical role of Nav1.8 in the pathogenesis of neuropathic pain has been well documented in rodents , 36. It SCN9A –23, whil Nav1.9) , and SCN Nav1.6) . Inherit Nav1.6) . It is n current , 32.+ channels such as Nav1.7 and “human pain in a dish”. hDRG neurons have been used to study acute inflammatory pain mechanisms following stimulation by inflammatory mediators such as prostaglandin E2 [In summary, naïve hDRG neurons provide a translational model in which to study voltage-gated Naandin E2 , 41. It andin E2 . Thus, n


The P2X7 receptor (P2X7R) is important in mediating a range of physiological functions and pathologies associated with tissue damage and inflammation and represents an attractive therapeutic target. However, in terms of their structure-function relationships, the mammalian P2X7Rs remain poorly characterised compared to some of their other P2XR counterparts. In this study, combining cysteine-based cross-linking and whole-cell patch-clamp recording, we examined six pairs of residues  located in different parts of the extracellular and transmembrane domains of the human P2X7R. These residues are predicted to undergo substantial movement during the transition of the receptor ion channel from the closed to the open state, predictions which are made based on structural homology models generated from the crystal structures of the zebrafish P2X4R. Our results provide evidence that among the six pairs of cysteine mutants, D48C/I133C and K81C/V304C formed disulphide bonds that impaired the channel gating to support the notion that such conformational changes, particularly those in the outer ends of the transmembrane domains, are critical for human P2X7R activation.The online version of this article (doi:10.1007/s11302-016-9553-0) contains supplementary material, which is available to authorized users. P2X receptors (P2XR) are a structurally distinctive family of ligand-gated ion channels activated upon extracellular ATP binding, which are expressed in many cell types and play a role in a wide range of physiological functions –4. TheseAR [V [The P2X7R, whilst exhibiting strong sequence similarity to other members of the P2X family and the conservation of key residues coordinating ATP binding, has unusual functional properties . For exaAR  and KV [AR [V ), subuniAR [V  and the AR [V , 21 and AR [V , P2X2R [AR [V  and P2X3AR [V . Here, wStructural models of the hP2X7R were produced based on the structure of the zfP2X4R in the closed and ATP-bound open states  using Modeller version 9.12  and analE. coli cells (Stratagene). Small-scale isolation of plasmid was performed using a mini-DNA preparation kit (QIAGEN). Mutations were confirmed by commercial sequencing (Beckman Coulter Genomics).Single and double cysteine mutations were introduced into the plasmid encoding the hP2X7R using a PCR-based site-directed mutagenesis method . In brie2, under humidified conditions. Cells were seeded in 6-well plates at 70–80% confluency prior to transfection and cells in each well were transfected using Lipofectamine2000 (Life Technologies) with 1 μg plasmid for the WT or mutant hP2X7R and 0.1 μg plasmid for enhanced green fluorescent protein (GFP), according to the manufacturer’s instructions.Human embryonic kidney (HEK) 293 cells were cultured in Dulbecco’s Modified Eagle Medium supplemented with 10% foetal bovine serum at 37 °C and 5% CO2, 2 mM CaCl2, 10 mM HEPES and 13 mM glucose, pH 7.3. Intracellular solution contained 145 mM NaCl, 10 mM EDTA and 10 mM HEPES, pH 7.3. Divalent cations strongly inhibit the P2X7R and therefore BzATP-induced currents were mainly measured in low divalent extracellular solution containing 147 mM NaCl, 2 mM KCl, 0.3 mM CaCl2, 10 mM HEPES and 22 mM glucose, pH 7.3. Three hundred micrometer BzATP was repeated applied for 4 s every 2 min, and when the currents were fully facilitated, cells were exposed to 10 mM DTT between BzATP applications.Cells were seeded onto 10-mm glass coverslips 20–24 h post transfection and single GFP-positive cells were chosen for recordings. Whole-cell currents were recorded at room temperature using an Axopatch 200B amplifier and analysed with pClamp 10.3 software (Axon instruments) as described in our previous studies , 32. Celt test for two groups and one-way analysis of variance test and Tukey’s post hoc test for more than two groups, and the difference was considered to be significant at p < 0.05.All results, where appropriately, are presented as the mean ± standard error of mean (SEM). Statistical analysis was carried out using Student’s Examination of the structural models of the hP2X7R in the closed and open states suggests that considerable conformational changes take place during receptor activation in the head, upper body and lower body of the extracellular domain as well as in the TM α-helices. Such conformational changes can be illustrated by the change in the distance, between the K81 and V304 (K81/V304) residues located in the upper body, I58/F311 and S60/L320 in the lower body, and A44/I331 and D48/I331 at the outer ends of the TM1 and TM2 domains  and also had no effect on BzATP-induced currents mediated by the WT receptor or single mutants Fig. a, b. LacAs introduced above, the P2X7R is physiologically and therapeutically important but our current understanding regarding its activation and the conformational changes which accommodate this has been mainly inferred by structural homology modelling and studies of single nucleotide polymorphic mutations . In thisIntroduction of single or double cysteine substitutions predominantly impaired or ablated receptor function Fig. c, d. ExpThe most interesting finding in this study is the contrast in results from the D48C/I331C and A44C/I331C double mutants. BzATP induced very small but discernible currents in cells expressing the D48C/I331C mutant Fig. c. FurtheThe upper body, based on comparison of the zfP2X4R structures in the closed and open states, remains much less mobile than other parts of the extracellular domain during receptor activation. In this study, we examined K81 and V304 in the upper body of the hP2X7R which are predicted to move significantly away from each other during receptor activation


The phase controlled synthesis of iron carbide nanoparticles was proposed through a thermodynamical and dynamical manner by introducing hetero-halide ions. 2C-hexagonal and monoclinic syngony, Fe5C2-monoclinic syngony and Fe3C-orthorhombic syngony) derived from body centered cubic Fe@Fe3O4 by introducing heteroatoms to restrain their phase transformation. We found that the phases of Fe2C NPs could be controlled by direct phase transformation in the drastic thermally driven procedure . Meanwhile, the selective adsorption of Cl ions weakened the bonding between Fe and C atoms, thus interfering with the penetration of C atoms to form lower carbon content Fe5C2 and Fe3C NPs. The kinetic mechanisms were evaluated using density functional theory (DFT) simulations focusing on the bonding energy between Fe–C and Fe–Cl atoms. All the obtained ICNPs exhibited typically soft ferromagnetic properties with the highest saturation magnetization value of 101.2 emu g–1 and the highest Curie temperature of 497.8 K.Iron carbide nanoparticles (ICNPs) are considered to have great potential in new energy conversion, nanomagnets and biomedical applications due to their intrinsically peculiar magnetic and catalytic properties. However, the synthetic routes were greatly limited in morphology and phase controlled synthesis. In this article, we present a versatile solution chemistry route towards colloidal ICNPs (Fe Among which, most work has been carried out on oxides and metallic iron. Although iron oxides are both air/water stable, and nontoxic in organisms, the saturation magnetization is low, especially at the nanoscale. Moreover, metallic iron nanostructures possess poor stability when exposed to an oxygen/water-containing atmosphere, which precludes their wider applications. Iron carbides (ICs), primarily consisting of carbon atoms occupying interstitial sites of the iron lattice, constitute an attractive alternative with high saturation magnetization (ca. 140 emu g–1), high chemical stability, and favourable bio-compatibility compared to oxides and metallic iron. Thus, ICs are promising multifunctional platforms for biomedical applications, such as bio-imaging, drug delivery, photo-thermal therapy and hyperthermia. In addition, ICs were reported to be potential magnetic storage media, and presented excellent catalytic activities in resource and electrochemical energy conversion, such as Fischer-Tropsch synthesis (FTS) and the oxygen reduction reaction.Iron based magnetic nanomaterials have gained intense interest both theoretically and practically in the last two decades, and are perfect alternatives for various platforms from biomedical applications to environmental catalysis. Solution chemistry routes, in which the generation and growth processes of particles are modulated by burst nucleation of the nucleus and precise supply of reactive entities, are important for the control of the size, shape, self-assembly and crystallinity of the nanostructures. Chaudret et al. reported the synthesis of near monodisperse Fe2.2C/Fe5C2 hybrid NPs utilizing seeded growth inspired by the FTS process. Besides, Fe3C NPs were also reported to be generated in ionic liquids [Cn-MIM][BF4]. Previously, our group reported the synthesis of single phase Fe5C2 NPs and evaluated their special catalytic activities in the FTS reaction. However, the phase and morphology control of ICNPs remains a great challenge due to the complicated phase diagram of Fe–C and uncontrolled growth.Despite the intriguing advantages of IC nanostructures, the synthetic routes were greatly limited in the morphology and phase controlled synthesis, which was probably due to their intrinsically ‘harsh’ properties. Until now, few reports have concerned their synthesis in the form of nanostructures. The major preparation routes of ICs could be generalized to solid state reactions, laser ablation methods, sono-chemical and bio-templated based methods, yet aggregations and polydisperse particles were unavoidable in the final products. Halide ions were demonstrated to influence the shapes of noble metal nanocrystals due to their varied binding energy on different metal facets. For another example, halide ions were also used in thermal decomposition of Fe(CO)5 to generate crystalline Fe NPs with average size distributions and robust stability in air, in which the strong bonding of Cl– and Fe induced the slow growth kinetics that was more thermodynamically stable. Despite all the distinct work reported, the function of halide ions was limited to the single-phase system. In fact, the peculiar selective abilities of halide ions to the metal surface make them a perfect platform for the modulation of ‘conversion chemistry’ from a special metal. How to introduce halide ions in the phase transformation system, and to explore their operational mechanism is interesting and significant.Generally, crystalline ICs are formed through the ‘penetration’ process of carbon atoms. The formation of ICs with different stoichiometry of Fe–C could be explained by their relative thermodynamic stability; exploring the process of their phase transformations in a both thermodynamically and kinetically controlled manner is important.2C , Fe5C2 (monoclinic syngony) and Fe3C (orthorhombic syngony) colloidal ICNPs could be obtained. Besides, the effects of halide ions on the formation process were simulated based on density functional theory (DFT). To our knowledge, it is the first time that ICNPs have been obtained in one system with tuned phases and growth kinetics.Herein, we report a facile and versatile solution chemistry synthesis of colloidal ICNPs of tuned phases and controlled morphologies by introducing hetero-halide ions. The synthetic procedures were conducted in a ‘seed-conversion’ manner; Fe5 in the presence of NH4Br produced spherical body centered cubic Fe (bcc-Fe) NPs with a homogeneous Fe3O4 shell, exhibiting a mean diameter of 14.0 ± 0.8 nm, as shown in 3O4 was confirmed by powder X-ray diffraction (PXRD) studies (see 3O4 were observed except for two strong peaks corresponding to the (110) and (200) facets of bcc-Fe, which was due to the peak broadening of their small and weak crystal domains of the Fe3O4 shell. The contrast in the high resolution transmission electron microscopy (HRTEM) image also revealed the core–shell structure  NPs. The shell was composed of amorphous entities with several tiny crystalline domains that represent an inverse spinel structured iron oxide Fe3O4. PXRD results carried out on powder samples showed the appearance of five peaks that confirm the carbon-rich IC phase hexa-Fe2C with the space group, P63/mmc (194). Interestingly, a void space clearly existed between the Fe2C core and Fe3O4 shell, which acted as the possible reactive area in the carbon penetration process, indicating the Kirkendall effect through the lattice transition. Besides, when the programmed temperature was higher than 290 °C, monoclinic carbon-rich carbide mono-Fe2C was obtained, with a diameter distribution of 15.0 ± 0.8 (2C (–101) and (–111) facets  shifted to small angles compared to hexa-Fe2C NPs (PDF#36-1249), indicating heavier lattice distortions compared to the initial bcc-Fe  symmetry, as shown in 2C could also be acquired when using trimethylamine-N-oxide (TMAO) as the oxidative agent to form Fe3O4 shells  image showed that the decomposition of Fe(CO)dies see . No peakture see . In the .0 ± 0.8  and expo.0 ± 0.8 . The PXR2C and inverse spinel structured hollow Fe3O4  and bcc-Fe (707.0 eV), compared to the h-Fe3O4 NPs, which was ascribed to the statistical Fe–C and metallic Fe bonding. The high-BE shift of metallic Fe to Fe–C bonding spectrum was probably due to the electrostatic interactions, spin–orbit coupling between the 2p core hole and unpaired 3d electrons of the photo-ionized Fe cations and Fe–C crystal field interactions. Besides, the Fe 2p3/2 spectrum could be well fitted using the Gupta–Sen multiplets for both the Fe2+ and Fe3+ components with a low-BE ‘pre-peak’ introduced for high-spin Fe2+ portions.A Shirley background-subtracted Fe 2p2 NPs, the near in situ products were examined using PXRD and GC-MS. PXRD patterns of the intermediate samples extracted at reaction temperature (Re. T) of 230 °C, 260 °C, 280 °C, and 310 °C are shown in 2C with (101) facets when the Re. T reached 260 °C . Then the main peak positions showed progressive shift to lower angles as the Re. T increased to higher than 260 °C, which was consistent with the expansion of the unit cell as the interstitial carbon atoms were incorporated  facet, to hexa-Fe2C, 2θ = 43.2°, (101) facet). The hexa-Fe2C NPs were fully formed at the Re. T of 280 °C. Interestingly, with the Re. T increased to 310 °C, mono-Fe2C NPs were obtained. The transformation was ascribed to a thermodynamic change with heavy lattice distortions due to a drastic thermally driven shift  and (–111) facets). To explore the transformation from hexa-Fe2C to mono-Fe2C NPs, we introduced PXRD characterizations of samples extracted at an average temperature interval of 15 °C, and further verified the gradual shifting process. Interestingly, no distinct crystal formation was observed upon increasing the Re. T to higher temperatures even at 345 °C, indicating that Fe2C phases were thermally favourable during the lattice transitions  scale" fill="currentColor" stroke="none"><path d="M0 1760 l0 -80 1360 0 1360 0 0 80 0 80 -1360 0 -1360 0 0 -80z M0 1280 l0 -80 1360 0 1360 0 0 80 0 80 -1360 0 -1360 0 0 -80z M0 800 l0 -80 1360 0 1360 0 0 80 0 80 -1360 0 -1360 0 0 -80z"/></g></svg>N. Moreover, in addition to 1-heptadecanenitrile and 1-hexadecanenitrile, plenty of saturated hydrocarbons such as heptadecane were detected which could be ascribed to the C–C cleavage of the nitriles. Meanwhile, the formed free C species acted as the carbon source. To verify the catalytic sites in the decomposition process, we synthesized h-Fe3O4 in the approximate size distribution of the Fe@Fe3O4 seeds, and conducted the synthetic process in the same procedure  and orthorhombic Fe3C (ortho-Fe3C) NPs were acquired through a ‘dynamically controlled’ manner. In the typical synthesis utilizing cetyltrimethyl ammonium chloride (CTAC) as the halide ion inducer, similar spherical ICNPs were obtained. TEM and HRTEM images of mono-Fe5C2 are shown in 3O4 shell. Interestingly, the ortho-Fe3C NPs (average diameter distribution of 14.1 ± 0.8 nm) could be obtained by increasing the additional halide ions systematically  and ortho-Fe3C , respectively, as shown in Different from the synthesis of hexa-Feally see . PXRD pr Besides, in a study by Sun et al., Cl ions were proposed as an agent that efficiently inhibited the Fe growth kinetics. To determine the roles that halide ions acted in the ICNPs formation process, we monitored the phase and morphology evolutions by tuning the reaction parameters systematically. By varying the additional amount of halide ions , the structures went through heavy lattice distortions from cubic to monoclinic and orthorhombic syngony. PXRD profiles of the performed particles showed that the crystalline phase was mono-Fe2C if without HIA-Cl. However, when the HIA-Cl was increased, the mono-Fe2C acted as the primary phase and gradually vanished with the appearance of the mono-Fe5C2 phase. The phase ratio of HIA-Cl-I to HIA-Cl-III was of 47% wt Fe2C : 53% wt Fe5C2, 17% wt Fe2C : 83% wt Fe5C2, and 57% wt Fe5C2 : 43% wt Fe3C, respectively. The complete disappearance of the mono-Fe2C phase initiated when the HIA-Cl was around 0.02 : 1, and finally transformed to the ortho-Fe3C phase when the HIA-Cl was around 0.04 : 1  or oleyl alcohol as co-surfactants were sufficient to avoid the collapse and crushing of the final products. This may arise from the robust chemical adsorption due to the strong affinity abilities of –P PBM data was replaced with SVG by xgml2pxml:<glyph-data id="z.dbd" format="PBM" resolution="300" x-size="34" y-size="34" xml:space="preserve"> 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000	</glyph-data><svg xmlns="http://www.w3.org/2000/svg" version="1.0" width="16.000000pt" height="16.000000pt" viewBox="0 0 16.000000 16.000000" preserveAspectRatio="xMidYMid meet"><metadata>Created by potrace 1.16, written by Peter Selinger 2001-2019</metadata><g transform="translate scale" fill="currentColor" stroke="none"><path d="M0 1440 l0 -80 1360 0 1360 0 0 80 0 80 -1360 0 -1360 0 0 -80z M0 960 l0 -80 1360 0 1360 0 0 80 0 80 -1360 0 -1360 0 0 -80z"/></g></svg>O and –OH to the Fe3O4 shell.Previously, Pd nanocubes with slight truncation at the corners and edges and Pd–Pt bimetallic nanocrystals were prepared using chemisorbed Br ions; the halide ions were introduced to ensure selective deposition of atoms onto the corner sites during seed-mediated growth. : 1 see . If the To verify the hypothesis above, we constructed a simulation model to evaluate the role Cl ions acted, focused on the affinity abilities. Based on the space symmetry of the Fe (101) surface, three highly possible adsorption configurations were considered for a single Cl(C) atom: configuration Cl(C)-I, where the Cl(C) atom was on the top-site of the Fe atom, configuration Cl(C)-II, where the Cl(C) atom was on the bridge-site of the Fe atoms, and configuration Cl(C)-III, where Cl(C) was on the hollow site of the Fe atoms see . GeometrWe then calculated the partial density of states (PDOS) of Fe atoms that bonded with the Cl(C) atom for the lowest energy configuration, as presented in 2C NPs; (2) a dynamically controlled manner, where the selectively adsorbed Cl ions weaken the bonding between Fe and C atoms, thus interfering with the absorption of C atoms, and forming lower carbon content Fe5C2 and Fe3C NPs.To examine whether the adsorption of Cl inhibited the adsorption of the C atom on the Fe surface, we introduced an additional C atom into the Cl(C) adsorbed Fe surfaces to explore the most preferable site of the second C atom. Starting with the most preferable configuration of the C(Cl) adsorbed Fe (101) surface, as shown in Cl(C)-III see , the secvia M–H loops with an applied magnetic field of 5 T and M–T curves in the temperature range 300–560 K. All the samples were purified and sealed in plastic capsules before characterization. The absolute magnetization value was deduced from Fe(0) elemental content determined using an inductively coupled plasma atomic emission spectrometer (ICP-AES). As was expected, all the initial bcc-Fe and performed ICNPs exhibited soft ferromagnetic behaviour at room temperature and 2 K, as shown in Ms and Hc values were slightly higher at 2 K than at 300 K, which could be interpreted by considering the thermal activation of the nano-structured materials. The measured saturation magnetization (Ms) of the initial bcc-Fe was 144.5 emu g–1, with a coercivity of 189.2 Oe, lower than the bulk entities (220 emu g–1), which was due to the size effect and the ferrimagnetic Fe3O4 shell. All the ICNPs exhibited lower Ms than that of the bcc-Fe, which was in accordance with the theoretical simulations that strong Fe–C bonding reduced the local magnetic moment of the Fe atoms.The magnetization measurements were investigated 3C NPs possessed the highest Ms values of the ICNPs, around 101.2 emu g–1, with a coercivity of 543.9 Oe, still lower than the bulk entities due to the size effects in the nanoscale. Interestingly, the hexa-Fe2C exhibited higher Ms values  than mono-Fe2C NPs . The mono-Fe5C2 possessed an Ms value of 95.0 emu g–1, and a coercivity of 636.5 Oe, which was lower than our previous report due to the smaller size. The M–T curves were measured using a vibrating sample magnetometer (VSM) with an applied probe field of 100 mT (see Tc) could be defined as the maximum slope in dM/dT deduced from a special field, as shown in Tc around 490 K, with the highest Tc of hexa-Fe2C (497.8 K), and the lowest Tc of ortho-Fe3C (477.8 K). The detailed magnetic measurement results are shown in The ortho-Fe0 mT see . The Cur2C) to monoclinic  and orthorhombic (Fe3C). It was the first time that ICNPs were obtained in one system with tuned phases and growth kinetics. The phase transformation mechanisms could be defined in a thermodynamically controlled and kinetically controlled manner. The selective adsorption of hetero-halide ions was essential in the synthesis of mono-Fe5C2 and ortho-Fe3C. Based on DFT simulations, we found that by selectively adsorbing Cl ions on the Fe surface, the C penetrating process was interfered with, thus restraining the additional C atoms into the Fe lattice to form lower carbon content ICNPs. All the preformed ICNPs expressed a typical soft ferromagnetic characterization with the highest Ms value of 101.2 emu g–1, and the highest Tc of 497.8 K. The proposed synthetic route based on the thermodynamic and kinetic control of the ‘seed-conversion’ method can be extended to other synthetic categories, especially in the synthesis of transition metal carbide nanostructures. Moreover, we present a generally new strategy to modulate the crystalline phase by introducing heteroatoms to restrain or accelerate the conversion process.In summary, we proposed a versatile solution method for the phase controlled synthesis of various colloidal ICNPs by introducing hetero-halide ions, with crystalline geometry from hexagonal 5 was injected to the reaction mixture and kept there for 30 min. A color change from salmon to brown then to black of the solvents were observed after ca. 1 min, indicating the decomposition of Fe(CO)5 and the formation of bcc-Fe NPs.In the typical synthesis, 62.5 mmol ODE, 0.1 mmol NHvia a syringe and the resultant solution was aged at 140 °C for another 30 min before it was cooled down to room temperature. Acetone (27 ml) was added to precipitate the product. After centrifugation , the product was collected and re-dispersed in hexane and precipitated using ethanol. The washing procedure was repeated three times and the final product, 14.0 ± 0.8 nm bcc-Fe NPs, was dispersed in methylbenzene with several droplets of OAm for further carbonization.To control the oxidation of the as-prepared bcc-Fe NPs, 1 mmol OA and hexane (0.2 ml) were added 2C NPs were synthesized as follows: ODA (37.5 mmol) or a mixture of ODA (11.5 mmol) and OAm (15.5 mmol) was magnetically blended in a four-neck flask and degassed under a gentle N2 flow for 1 h at 120 °C. Then, the resulted bcc-Fe NPs  were added via a syringe and the reaction solution was heated at 130 °C for another 30 min to remove hexane thoroughly before it was heated to the target temperature (260–345 °C) for 15–45 min. The black-brown colored solution was cooled down to room temperature by removing the heating source. Acetone (27 ml) was added and the mixture was centrifuged . The precipitates were purified with hexane (15 ml) and ethanol (20 ml) three times before being dispersed in methylbenzene (10 ml) with several droplets of OAm. Fe5C2 and Fe3C NPs were obtained in the same procedure except for the addition of halide entities (CTAC or NH4Br) and TOPO (0.05–0.07 mmol), or oleyl alcohol (8–12 mmol): Fe5C2 NPs–NH4Br (0.03–0.05 mmol), or CTAC (0.11–0.13 mmol); Fe3C NPs–NH4Cl (0.11–0.13 mmol), CTAC (0.17–0.21 mmol).Typically, FeAb initio Simulation Package (VASP). The exchange-correction potential was treated by using the generalized gradient approximation (GGA) in the form proposed by Perdew, Burke, and Ernzerhof (PBE). A plane-wave basis set with the projector augmented plane-wave (PAW) method was used to calculate the total energies and forces, and to optimize the geometries. We have modeled the surface of bcc-Fe having the (101) orientation by a slab consisting of five atomic layers with a (2 × 2) supercell containing 40 Fe atoms in the A-B-A-B-A stacking pattern. Each slab was separated from the other by a vacuum region of 15 Å, and the iron atoms on the bottom three layers were held at their bulk configuration while those on the upper two layers were allowed to relax without any symmetry constraint. The reciprocal spaces were represented by Monkhorst–Pack special k-points of 7 × 7 × 1 meshes. The 2s22p2, 3s23p5, and 3d64s2 atomic orbitals were treated as the valence states for C, Cl, and Fe, respectively. Because the GGA could not properly describe the strongly correlated system containing partially filled d atoms, we used the GGA + U method by dividing the electrons into two classes: delocalized s and p electrons, which could be well described by the GGA, while the localized d electrons were described by the Coulomb and exchange corrections. We used a correlation energy (U) of 4.00 eV and an exchange energy (J) of 1.00 eV for Fe 3d orbitals. These values have been tested and widely used in previous studies. The energy cut-off was set to 500 eV, and the convergence in energy and force was 10–4 eV and 10–2 eV Å–1, respectively. Bader charge analysis was carried out to study the charge distribution and transfer quantitatively.Our first principles calculations within the framework of density functional theory (DFT) were carried out using Vienna


Cabazitaxel has not been studied in patients with hepatic impairment (HI). This phase I study assessed cabazitaxel safety and pharmacokinetics in patients with HI.2, respectively. Doses were escalated in patients with HI based on Cycle 1 dose-limiting toxicities (DLTs). Adverse events and the cabazitaxel pharmacokinetic profile were assessed.Patients with advanced, non-hematologic cancer, and normal hepatic function (Cohort 1: C-1), or mild (C-2), moderate (C-3), severe (C-4) HI received cabazitaxel starting doses of 25, 20, 10, and 10 mg/m2 experienced DLTs; maximum tolerated dose (MTD) was 20 mg/m2. In C-3, two patients receiving 20 mg/m2 experienced DLTs; MTD was 15 mg/m2. C-4 was discontinued early due to DLTs. The most frequent cabazitaxel-related, grade 3–4 toxicity was neutropenia (42%). Cabazitaxel clearance normalized to body surface area (CL/BSA) was lower in C-1 (geometric mean [GM] 13.4 L/h/m2) than expected (26.4 L/h/m2), but similar in C-2 (23.5 L/h/m2) and C-3 (27.9 L/h/m2). CL/BSA in C-4 was 18.1 L/h/m2. Compared with C-2, CL/BSA increased 19% in C-3 , but decreased 23% in C-4 . Cabazitaxel free fraction was unaltered. No significant correlation was found between grade 3–4 toxicities and pharmacokinetic parameters.In C-2, three patients receiving cabazitaxel 25 mg/m Mild–moderate HI did not cause substantial decline in cabazitaxel clearance. Cabazitaxel dose reductions in patients with mild–moderate HI, and a contraindication in patients with severe HI, are justified based on safety data. Cabazitaxel, a second-generation semisynthetic taxane, has demonstrated activity in the second-line treatment of metastatic castration-resistant prostate cancer (mCRPC) after progression on docetaxel-based treatment . CabazitHepatic impairment may have an unpredictable impact on the pharmacokinetics (PK) of chemotherapies metabolized by the liver, and low serum albumin levels associated with hepatic impairment can result in an increased fraction of free drug leading to increased toxicity –9. BasedAs previous studies of cabazitaxel in solid tumors excluded patients with hepatic impairment, the safety profile of cabazitaxel in this subgroup has not been established , 10. HerThis was an open-label, dose-escalation, multicenter, phase I study NCT01140607) of cabazitaxel in patients with non-hematologic cancers and varying degrees of hepatic function. This study was designed to evaluate the maximum tolerated dose (MTD) and safety, and assess the PK properties and relationship between PK and safety parameters, of cabazitaxel in patients with varying degrees of hepatic impairment. A similar design was employed in the study of irinotecan in patients with hepatic dysfunction [40607 of Eligible patients were aged ≥18 years with a life expectancy of >3 months, diagnosed with metastatic or locally advanced non-hematologic cancer for which no effective curative therapy was available, had refractory or progressive disease following standard therapies, and had normal hepatic function or chronic hepatic impairment. Patients were enrolled into one of four cohorts based on their degree of hepatic function, defined using National Cancer Institute (NCI) criteria . Cohort 2, Cohort 2 (mild hepatic impairment) received 20 mg/m2, and Cohorts 3 and 4 (moderate or severe hepatic impairment) received 10 mg/m2. The starting dose for Cohort 4 was decided by the Study Committee based on safety and PK findings in the first three patients treated in Cohort 3. Doses were adjusted in Cohorts 2, 3, and 4 based on DLTs observed in Cycle 1. In Cohort 2, doses were adjusted to 15 mg/m2 (DL –1) or 25 mg/m2 (DL +1), and in Cohorts 3 and 4, doses were adjusted to 15 mg/m2 (DL +1), 20 mg/m2 (DL +2), or 25 mg/m2 (DL +3). If a Cycle 1 DLT was observed in at least two of up to six patients at a given dose level, no further dose escalation occurred. If a Cycle 1 DLT occurred in one of the first three patients treated at a given dose level, three additional patients received that dose. The MTD was the highest dose at which none of the first three patients or one of up to six total patients experienced a Cycle 1 DLT up to the 25 mg/m2 dose. Use of granulocyte colony-stimulating factor (G-CSF) was not permitted during Cycle 1.Patients received cabazitaxel during a 1-hour intravenous (IV) infusion on Day 1 of each 3-week cycle until unacceptable toxicity, disease progression, withdrawal of consent, investigator decision, or study cutoff. Different starting doses of cabazitaxel were used for each cohort based on information from the BEX6702 study . The cabSafety evaluations included vital signs, physical examinations, ECOG performance status, electrocardiograms, and laboratory parameter tests. Adverse events (AEs) were assessed according to the NCI Common Terminology Criteria for AEs (CTCAE) v4.03  from themax), area under the concentration versus time curve extrapolated to infinity (AUCinf), area under the concentration versus time curve calculated using the trapezoidal method from time 0 to real-time tlast (AUClast), terminal half-life (t1/2z), total body clearance (CL), and volume of distribution at steady state (Vss). CL and Vss were normalized to body surface area . Effect of hepatic impairment on cabazitaxel PK parameters  was evaluated using linear mixed-effect modelling with degree of hepatic impairment as the fixed effect. Additional plasma samples were collected 5 min before, 3 h after and 24 h after the start of infusion to determine the cabazitaxel free fraction after equilibrium dialysis in buffer using a validated LC-MS/MS method with a LLOQ of 0.1 ng/mL. Cabazitaxel free fraction was estimated using a linear mixed-effect model, with cohort and dose level as fixed effect and time and BSA as continuous variable and patient as random effect.Heparinized blood samples were collected from all patients in Cycle 1, on Day 1 immediately prior to the start of infusion, 5 min before the end of infusion, and then at 5, 15, and 30 min and 1, 2, 3, 5, 7, and 10 h after the end of infusion. Samples were also collected on Days 2, 3, 4, 5, 8, and 10 after cabazitaxel infusion. Cabazitaxel plasma concentrations were analyzed using a validated liquid chromatography with tandem mass spectrometry method . Cabazitaxel PK parameters were calculated using non-compartmental analysis with validated software . PK parameters included maximum observed concentration (Cn = 34) were considered non-eligible based on inclusion and/or exclusion criteria. Overall, approximately half of the patients were male, median age was 60 years (range 18–79 years), and most patients (81%) had an ECOG performance status of 1. Patients had various primary tumors with colon and liver the most frequent (19% each). At study entry, most patients had metastatic disease (91%), some had locally advanced disease (7%), and a minority had locoregional recurrence (2%). Median time from cancer diagnosis to first cabazitaxel dose was 2.93 years (range 0.5–17.9 years), and median time from last relapse/progression to first cabazitaxel dose was 1.08 months (range 0.2–24.2 months). Approximately two-thirds of patients had received three or more prior anticancer regimens.Of 77 patients screened, 43 were enrolled including six patients in Cohort 1 , 18 in Cohort 2 (mild impairment), 12 in Cohort 3 (moderate impairment), and seven in Cohort 4 (severe impairment) , 20 and 25 mg/m2; and Cohorts 3 (moderate impairment) and 4 (severe impairment), 10, 15, and 20 mg/m2 (Table 2) who had received 31 cycles. Primary reasons for cabazitaxel discontinuation included disease progression (65%) and toxicity/AEs (21%) , 25 mg/m2 experienced DLTs , and the MTD was established as 20 mg/m2. In Cohort 3 (moderate impairment), the first two patients treated at 20 mg/m2 experienced DLTs , and the MTD was established as 15 mg/m2. In Cohort 4 (severe impairment), the MTD was not established because no patient treated with 10 or 15 mg/m2 experienced DLTs during Cycle 1 and treatment was discontinued early for this cohort because the first patient treated at 20 mg/m2 experienced DLTs and subsequently died from a combination of septic shock, tumor lysis syndrome and acute respiratory failure in the context of acute renal failure and disease progression. Based on this outcome, patient accrual into Cohort 4 was discontinued.Of the 43 treated patients, 38 were evaluable for DLTs with five excluded due to concomitant G-CSF administration during Cycle 1 in the absence of a DLT. In Cycle 1, 13 patients (34%) across all cohorts experienced a DLT  that was approximately 20-fold higher than other patients in Cohort 3 (30.5 L/h/m2 for patients receiving cabazitaxel 15 and 20 mg/m2 collectively). The CL/BSA estimate for patients in Cohort 1  was in the very low range of typical cabazitaxel clearance shown in a previous population PK analysis  [2, n = 4 [2, n = 25 [2n = 21 [last/dose, whereas Cohort 4 (severe impairment) showed a 23% decrease in CL/BSA, associated with a 17% increase in AUClast/dose  was found between grade 3–4 TEAEs or laboratory abnormalities in Cycle 1 and PK parameters. Study cohort (degree of hepatic function) was not a statistically significant covariate in any of the logistic regression models of PK parameters.No significant correlation  and disease progression; therefore, the MTD in this cohort of patients remains undetermined.The MTD of cabazitaxel administered by IV infusion every 3 weeks in patients with advanced solid tumors was determined to be 20 and 15 mg/m2 (MTD) in patients with mild or moderate hepatic impairment, respectively, was generally similar to that observed in patients with normal hepatic function receiving 25 mg/m2. In this study, prophylactic use of G-CSF was not permitted during Cycle 1. Prophylactic administration of G-CSF has the ability to reduce hematologic toxicity in clinical practice.The overall safety profile of cabazitaxel was consistent with the known safety profile with no new safety issues identified. The safety profile of cabazitaxel 20 and 15 mg/mn = 6), which may help explain the large variability in cabazitaxel clearance for this cohort as several outliers considerably affected the average values of the cohort and created a substantial shift in average CL/BSA. Patients with mild or moderate hepatic impairment had CL/BSA values comparable to historical data, suggesting no influence of mild or moderate hepatic impairment on cabazitaxel PK. There was no evidence that moderate versus mild hepatic impairment resulted in a substantial decline in cabazitaxel clearance. There was no evidence that the lower MTD in patients with mild or moderate hepatic impairment, compared with the approved 25 mg/m2 cabazitaxel dose, were due to higher cabazitaxel exposure. Patients with severe hepatic impairment had a numerically decreased CL/BSA compared with mildly impaired patients, indicating some effect of severe impairment on PK parameters. This numerical increase in cabazitaxel exposure may, in part, explain the increased toxicity of cabazitaxel observed in this patient cohort. However, because of the limitations of a small study and unbalanced sample sizes per cohort, this observation should be interpreted with caution.As CL/BSA for cabazitaxel in patients with normal hepatic function was low compared with historical data , 15–17, Hepatic impairment had no effect on the cabazitaxel unbound fraction with a low free fraction estimated across all cohorts (5.6–6.6%). These results are consistent with the high binding of cabazitaxel to total plasma proteins observed ex vivo and in vitro 89–92%) [% 2, 15,,15, 17.2, respectively, compared with the approved dose of 25 mg/m2. Based on this study and in the absence of appropriate data, the use of cabazitaxel is not recommended in patients with severe hepatic impairment. Based on PK data, there was no evidence of a relationship between safety and PK parameters as the lower MTDs could not be justified by higher cabazitaxel exposure in patients with mild or moderate hepatic impairment. However, dose reductions of cabazitaxel in patients with mild or moderate hepatic impairment, and contraindication in patients with severe hepatic impairment, are justified based on safety data. A recent phase III non-inferiority study (PROSELICA) has demonstrated that cabazitaxel administered at a dose of 20 mg/m2 maintains at least 50% of the survival benefit observed with the approved 25 mg/m2 dose of cabazitaxel versus mitoxantrone in the previous phase III TROPIC study [Even though cabazitaxel is primarily metabolized by CYP3A in the liver, the minimal impact of hepatic impairment on cabazitaxel PK parameters is consistent with a high cabazitaxel clearance driven by hepatic blood flow and is also consistent with the modest effect that repeated ketoconazole (a strong CYP3A inhibitor) administration has on cabazitaxel clearance; in one study, repeated ketoconazole administration resulted in a 20% decrease in cabazitaxel clearance [IC study , in patiIC study .