Background and objectives: The incidence of cutaneous melanoma has been increasing. Melanoma is an aggressive form of skin cancer irresponsive to radiation and chemotherapy, rendering this cancer a disease with poor prognosis: In order to surpass some of the limitations addressed to melanoma treatment, alternatives like vitamins have been investigated. In the present study, we address this relationship and investigate the possible role of vitamin A. Materials and Methods: We perform a co-culture assay using a macrophage cell model and RAW 264.7 from mouse, and also a murine melanoma cell line B16-F10. Macrophages were stimulated with both Escherichia coli lipopolysaccharides (LPS) as control, and also with LPS plus vitamin A. Results: Using B16-F10 and RAW 264.7 cell lines, we were able to demonstrate that low concentrations of vitamin A increase cytotoxic activity of macrophages, whereas higher concentrations have the opposite effect. Conclusion: These findings can constitute a new point of view related to immunostimulation by nutrients, which may be considered one major preventive strategy by enhancing the natural defense system of the body. Skin cancers represent a major public health problem ,2. The iThere are some established risk factors for melanoma development, like family history ,10, geneVitamin A can be highlighted due to its role in normal and cancer conditions ,12,13. VIt could also be discussed if vitamin A active metabolites play some role in melanogenesis, but still this issue has not yet been elucidated ,17. The There are some evidences indicating the benefits of vitamin A in cancer therapy. However, the use of vitamin A for both chemoprevention or even for therapeutic applications remains controversial ,14. AccoThe mouse melanoma B16-F10 cells exhibit in vitro a mixed morphology of spindle-shaped and epithelial-like cells. It was originally isolated from C57BL/6J mouse strain affected with melanoma. B16-F10 is a metastatic variant of B16 melanomas with high tropism for lung invasion.The mouse RAW 264.7 cells exhibit in vitro a morphology of monocyte/macrophage cells and were originally isolated from Abelson murine leukemia virus-induced tumor of the BALB/c mouse strain.2. B16-F10 (104 cells/mL) and RAW 264.7 (2 × 104 cells/mL) were seeded in 96-wells plates and allowed to adhere for 24 h. Unless otherwise specified, all treatments and controls were carried out in serum-free conditions [B16-F10 murine melanoma cell line (ATCC CRL-6475) and RAW 264.7 macrophage-like cell line (ATCC TIB-71) were maintained in Dulbecco’s modified eagle’s medium . The media formulation was supplemented with 10% heat-inactivated fetal bovine serum  and 1% penicillin/streptomycin/amphotericin B mixture (Sigma-Aldrich). Cells were grown at 37 °C under a humidified atmosphere with 5% CO2O2) concentration to induce oxidative stress [2O2 concentrations  for 24 h. The experimental concentration was determined by thiobarbituric acid reactive substances (TBARS) assay, and cell metabolic activity was measured by 3--2,5-diphenyltetrazolium bromide (MTT) assay. Cells incubated with serum-free medium were considered the controls to this experiment (results not shown).To determine the accurate hydrogen peroxide  were seeded in 24-wells plates and allowed to adhere for 24 h. Afterwards, melanoma cells were cultured with standard H2O2 treatments in serum-free conditions for 24 h. Then 50% trichloroacetic acid  was added to each treatment. The mixture was centrifuged  and 1% thiobarbituric acid  was added to the supernatant that was maintained in the water-bath for 40 min. The detection was performed using the Jenway 6405 UV/Vis spectrophotometer  at wavelength absorbance of 535 nm (results not shown).B16-F10 cells  for 24 h. Cell metabolic activity was measured by MTT assay and the experimental concentrations were defined as the lowest value that does not considerably alter the cell viability, and the highest value that considerably alters the cell viability. Cells incubated with serum-free medium were considered the controls for this experiment (results not shown).2O2 treatment (25 µM) for 24 h. Then B16-F10 cells were submitted to two different concentrations of vitamin A (0.1 and 100.0 µg/mL) for 24 h. Cell metabolic activity was measured by MTT assay. Cells incubated with serum-free medium were considered the controls for this experiment. To determine the possible redox effect of vitamin A, B16-F10 malignant melanocytes were previously submitted to a H®  microplate reader  [To measure B16-F10 and RAW 264.7 cell activity, after a 24 h incubation with different treatments, 20 µL of the 3--2,5-diphenyltetrazolium (MTT) (Sigma–Aldrich) was added into each 96-plate well, followed by a 3 h incubation period. Assays using tetrazolium salts are generally used to evaluate proliferation and cell viability. Cell proliferation is defined as the cellular growth rate and cell viability determined by the number of live cells, and is usually expressed as a percentage of the control . The detion 2.6) .Escherichia coli O111:B4  1000 ng/mL. The activated macrophages were co-incubated with B16-F10 melanoma cells (10:1 ratio) at 37 °C in a 5% CO2 incubator for 24 h. Then cells were treated with different concentrations of vitamin A (0.1 and 100.0 µg/mL), and the cytotoxic effects of LPS-activated macrophages–melanoma cells were measured, calculated, and expressed as follows, were OD stands for optic density for the MTT assay [RAW 264.7 macrophage-like cells were incubated with lipopolyssacharides (LPS) from TT assay :% Cytot2O2 and vitamin A treatments), and with Tukey’s multiple comparisons test for comparisons between every different treatment/condition. Significance was set at p < 0.05.The variables were expressed as mean ± SD (standard deviation). Data were analyzed with GraphPad Prism 6.0 . When three or more conditions were evaluated, statistical analysis was conducted through one-way ANOVA with Sidak post hoc test for comparisons between control and treatments (HAfter the establishment of a dose versus response curve for the assessment of vitamin A effects (results not shown), the possible redox effects of the concentration of 0.1 µg/mL were studied. These results are shown in 2O2 treated cells, as well as vitamin A treated cells and H2O2 + vitamin A treated cells. Metabolic activity of cells allowed us to study viability of the cells on different conditions of treatments. All of these comparisons are significant. H2O2 treatments significantly reduce the metabolic activity of melanoma cells (p < 0.0001). The same evidence is found in vitamin A treatment (p < 0.05), and in the combined treatment of H2O2 + vitamin A (p < 0.05).2O2. Results demonstrate that 0.1 µg/mL vitamin A may buffer the effect caused by H2O2 (p < 0.05), since there is no significant difference between cells treated with vitamin A alone and those treated with the vitamin and the same amount of H2O2 (25 µM) that significantly decreases metabolic activity. The possible redox effects of the concentration of 100 µg/mL were also studied. These results are shown in 2O2, to vitamin A, and to H2O2 + vitamin A treated cells. All of these comparisons are significant. H2O2 (25 µM) treatments significantly reduce the metabolic activity of melanoma cells (p < 0.0001), as well as 100 µg/mL vitamin A (p < 0.0001) and H2O2 + 100 µg/mL vitamin A treatments (p < 0.0001). 2O2 treated cells to vitamin A treated cells and to H2O2 + vitamin A treated cells. All the comparisons are significant.2O2 treated cells to cells treated with 100 µg/mL vitamin A, it is possible to observe an even more significant decrease (p < 0.0001) in the metabolic activity of the tumor cells. Additionally, comparing the effect of 100 µg/mL vitamin A treatment in cells previously submitted to 25 µM H2O2, not only the buffering effect of 0.1 µg/mL vitamin A was not observed. Contrariwise, an even lower metabolic activity was found (p < 0.0001) in melanoma cells than the metabolic activity achieved with both treatments (25 µM H2O2 and 100 µg/mL vitamin A) alone. When comparing HFor the assessment of macrophage cytotoxicity, RAW 264.7 cells were previously incubated with 1 µg/mL LPS. After 24 h, cells were also incubated with 0.1 and 100.0 µg/mL vitamin A concentrations. After LPS activation, two groups were treated with the two different concentrations of vitamin A previously used. According to the cytotoxic algorithm created by Seo and collaborators , the perDespite all the knowledge about melanoma biology, this cancer remains one of the greatest challenges of our day ,5,32,33,Although the diagnosis is relatively easy, there is an emerging need for new compounds capable of preventive or curative properties. That way, there are growing fields that state the possible role of dietary factors ,11, as wEmerging evidence indicates that dietary factors, such as antioxidant compounds, could have a double role, acting as an antioxidant and pro-oxidant, depending on the concentrations used ,35,36. S2O2 and vitamin A treatments alone are capable of reducing cell metabolic activity in a significant way. When treatments are combined, 0.1 µg/mL vitamin A has the capability of an increase in cell metabolic activity that surpasses the percentage of metabolic activity of H2O2 treatment alone , whereas 100 µg/mL vitamin A treatment presented a cytotoxicity of only 75.7%. Thus, activated macrophages stimulated with 100 µg/mL vitamin A treatment presented a slight increased cytotoxicity of only 1.9%.These results show the capability of lower concentrations of vitamin A to increase the macrophage activity more effectively than higher concentrations of vitamin A treatment, thus enhancing macrophage-induced cytotoxicity.If this work ended at the redox studies, one may suggest that vitamin A could have a possible antitumor effect/role ,35,36. HThe discussion of the effect of vitamins in the literature is almost inconclusive, since there are no studies devoted to the investigation of the dose–response effect of those vitamins. Although these results are encouraging, new questions are now evolving. Given that there is little information about vitamin A and melanoma, further studies should be done in order to validate the results presented in this study.


Drosophila males. We found that mild activation of P1 neurons was sufficient to affect sleep, but not courtship or feeding, while stronger activation of P1 neurons labeled by four out of five independent drivers induced courtship, but only the driver that targeted the largest number of P1 neurons affected feeding. These results reveal a common neural node that affects sleep, courtship, and feeding in a threshold-dependent manner, and provide insights into how competing behaviors can be regulated by a shared neural node.Animals choose among sleep, courtship, and feeding behaviors based on the integration of both external sensory cues and internal states; such choices are essential for survival and reproduction. These competing behaviors are closely related and controlled by distinct neural circuits, but whether they are also regulated by shared neural nodes is unclear. Here, we investigated how a set of male-specific P1 neurons controls sleep, courtship, and feeding behaviors in The online version of this article (10.1007/s12264-018-0281-z) contains supplementary material, which is available to authorized users. Drosophila melanogaster as a model system using genetic and physiological approaches makes it ideal for exploring the neural mechanisms underlying a variety of behaviors including sleep, courtship, and feeding. Indeed, substantial progress has been made on how these individual behaviors are controlled by specific neuronal circuits, often referred to as sleep circuits, courtship circuits, and feeding circuits [A fundamental question in biology is how animals sense environmental cues and alter their physiology and behavior in ways that are beneficial for their survival and reproduction. The amenability of circuits –6.Sleep, courtship, and feeding behaviors are mutually exclusive in principle. Very little is known about how these behaviors cross-talk at the level of neuronal circuits. Recently, we showed that sleep and sexual behaviors interact in a sex-specific way and identified sexually dimorphic neurons that mediate the interplay between sleep and sex . Among tDrosophila.Here, we used five independent driver lines that labeled P1 neurons ranging from 9 to 23 cells per hemisphere  to determine how P1 activation affects sleep, courtship, and feeding behaviors in Split-GAL4 reagents including R15A01-AD, R17D06-AD, R71G01-DBD, and R22D03-DBD, as well as R17D06-LexA and R71G01-LexA have been described previously [dsxGAL4, UAS>stop>dTrpA1, UAS>stop>myrGFP, and LexAop2-FlpL were used as previously described [Flies were maintained at 22°C under a 12 h:12 h light:dark cycle. eviously , 22 and escribed .Individual 2–4 day-old male flies were placed in locomotor activity monitor tubes  with fly food (2% agarose and 5% sugar), and entrained under 22°C and 12 h:12 h light:dark conditions for at least 2 days before sleep tests. One day of sleep data were first recorded at 22°C as baseline, then the flies were shifted to 25.5°C, 27°C, 28.5°C, or 30°C for two days, and then returned to 22°C for at least one day. Sleep was analyzed as previously described [We used unilateral wing extension in isolated males to compare courtship induced by P1 activation. Males were individually placed in 2 cm-diameter round chambers with food (2% agarose and 5% sugar) at 25.5°C, 27°C, 28.5°C, or 30°C and video was captured for 24 h, starting at 09:00. We then manually scored the percentage of males displaying unilateral wing extension. The average walking velocity during the 24-h recording was further quantified using the ZebraLab software system .dTrpA1 activation. Thereafter, they were transferred to 1% FD&C Blue 1  food  for 15 min (between 15:00 and 17:00) at the above temperatures for continuous activation while allowing feeding. To quantify the food intake, the absorbance of the ingested blue dye was measured as previously described [Feeding was assayed using food with blue dye. In brief, flies were starved for 24 h on 1% aqueous agarose at 22°C, then moved to 25.5°C, 27°C, 28.5°C, or 30°C for 30 min for We dissected the brains of 4–6 day-old male or female flies in Schneider’s insect medium  and fixed them in 4% paraformaldehyde in phosphate-buffered saline (PBS) for 30 min at room temperature. After 4 × 15-min washes in PAT , tissues were blocked in 3% normal goat serum (NGS) for 60 min, then incubated in primary antibodies diluted in 3% NGS for ~24 h at 4°C, washed (4 × 15-min) in PAT, and incubated in secondary antibodies diluted in 3% NGS for ~24 h at 4°C. Tissues were then washed (4 × 15-min) in PAT and mounted in Vectashield  for imaging. The primary antibodies used were rabbit anti-GFP  and mouse anti-Bruchpilot . The secondary antibodies used were goat anti-mouse IgG conjugated to Alexa 555  and goat anti-rabbit IgG conjugated to Alexa 488 . Samples were imaged at 20× magnification on a Zeiss 700 confocal microscope, and processed with ImageJ.Statistical analysis was performed using Prism GraphPad as indicated in the figure legends.GAL4 drivers for labeling and manipulating P1 neurons. We previously used two intersectional methods to target P1 neurons, one using the split-GAL4 system  that also label male-specific P1 neurons and thus made three additional splitGAL4s . The four split-GAL4s each labeled 9–12 P1 neurons  to investigate how the activation of these neurons via the temperature-sensitive activator dTrpA1 [a, P1b, P1c, and P1e, but not P1d neurons significantly inhibited sleep  was able to induce unilateral wing extension . The P1e driver labeled ~23 P1 neurons, while the other four P1 drivers each labeled 9–12 P1 neurons. There are at least two possibilities why only activation of P1e but not P1a–P1d neurons suppressed feeding behaviors in starved males: (1) P1e neurons include a set of neurons that are not labeled by other P1 drivers, and these neurons specifically affect feeding behavior; or (2) as the number of P1 neurons labeled by P1e is about double that of P1a–P1d, it is possible that feeding requires the activation of a larger number of P1 neurons than sleep or courtship. Together, these results indicate that sleep, courtship, and feeding behaviors are all affected by activation of P1 neurons, but with different activation thresholds.We next used these five P1 drivers . The two control lines also showed a slightly increased walking velocity at 30°C. As only activation of P1e neurons, but not P1a–P1d, suppressed feeding, this suppression could not be due to increased locomotion. To further test if feeding and walking velocity were negatively correlated, we plotted all the feeding and locomotor data for all P1-activated males and control males at 25.5°C, 27°C, 28.5°C, and 30°C, and found that feeding and walking velocity were not negatively correlated, and even slightly positively correlated (r = 0.37), although the correlation was not significant (P = 0.0503). These results indicate that decreased feeding by P1e activation is not due to increased locomotion. How P1e neurons regulate feeding is still unclear and awaits further investigation.We next asked whether the suppression of feeding by activation of P1velocity . We assae.g., P1e), suggesting that P1 activity affects all sleep, courtship, and feeding behaviors. Furthermore, we found that differential activation thresholds for P1 neurons were required to affect these three behaviors  of these targeted P1 neurons affect sleep, courtship, and feeding behaviors. We found that all these behaviors were affected by stronger activation of at least one P1 driver  that participates in all these competing behaviors, but also how P1 neurons modulate these behaviors in a hierarchical manner Fig. B.There are nearly 50 pairs of P1 neurons in the male fly brain , and we e.g., different amounts of sleep or feeding) are relatively smaller and underestimated, and the mechanism underlying these differences is unclear. That P1 neurons are male-specific and regulate sleep, courtship, aggression, and feeding suggests that sexual dimorphism in these behaviors may be greater than we thought, and our results provide a simple model of how a small number of sex-specific neurons can contribute to various sexually-dimorphic behaviors.We also note that, although males and females play distinct roles in sexual behavior, their differences in non-sexual behaviors Below is the link to the electronic supplementary material.


Yarrowia lipolytica, which is model oleaginous yeast with high industrial interest, was cultivated on fatty substrates. Data concerning fatty acid composition of both substrate and yeast lipids and comparisons of the experimental data with model predictions presented in “Biomodification of fats and oils and scenarios of adding value on renewable fatty materials through microbial fermentations: Modelling and trials with Yarrowia lipolytica”  were provided. Furthermore, the total yeast lipids were fractionated into their main fractions, that is, phospholipids, glucolipids plus sphingolipids and neutral lipids, and the fatty acid composition of each lipid fraction was reported. Specifications tableValue of the data•Yarrowia lipolytica.The data can be used in order to identify the fatty acid specificity of •Y. lipolytica can be pre-determined.The composition of lipids  accumulated in •New biomodification processes of common fats can be designed.1Yarrowia lipolytica growing on olive oil, linseed oil, palm oil, sunflower oil, cod liver oil, and beef tallow, and two Figures showing: (1) Experimental data and theoretical predictions of the fatty acid composition of extracellular and intracellular lipids of Y. lipolytica and (2) theoretical fatty acid profiles of the free fatty acid fraction released in the growth medium during growth of Y. lipolytica on the above mentioned fatty substrates.The data article includes 2Y. lipolytica ACA-DC 50109 was used in the current investigation. The strain was maintained on potato dextrose agar  at 7 ± 1 °C and re-cultured twice a month.The yeast 4.7H2O , 1.5; KH2PO4 (Fluka), 7.0; Na2PO4 (Fluka), 2.0; CaCl2.2H2O , 0.1; ΖnSO4.7H2O , 0.001; CuSO4.5H2O , 0.0001; Co(NO3)3.3H2O (Merck), 0.0001; MnSO4.5H2O (Fluka), 0.0001; (NH4)2SO4 (Fluka), 0.5; yeast extract , 2.0. Various commercial fats of plant  and animal  origin were used as carbon and energy sources at a concentration of 25 g/L.The growth media contained (in g/L): MgSO6 cells/mL. The cultures were incubated in a rotary shaker  at 180 rpm and T = 28 ± 1 °C.Experiments were performed in 250-mL Erlenmeyer flasks. The flasks containing 50±1 mL of growth media were sterilized at 121 °C for 20 min and thereafter inoculated with 1 mL of a mid-exponential phase pre-culture containing 4×10Determination of extracellular and intracellular lipids was performed as described in The predictions have been obtained using the mathematical model which is presented in Experiments were performed in duplicate. Data represent means of two replicates. , Fig. 2F


Background Like immune cells, platelets express the repertoire of toll-like receptors (TLR), among them TLR2 and TLR4, which are important for the recognition of bacterial patterns. Receptor-mediated functional effects in platelets have been investigated, but reliable conclusions are tampered due to heterogeneous study designs with variable platelet preparation methods. This study compares TLR2- and TLR4-dependent platelet responsiveness in platelet-rich plasma (PRP) and in washed platelets (WPs).Material and Methods Fresh peripheral blood samples from healthy donors served for the preparation of PRP and WP. Basal and agonist-stimulated TLR2 and TLR4 expression levels were evaluated by flow cytometry. Light transmission aggregometry was used to investigate functional effects of TLR2 and TLR4 stimulation with Pam3CSK4 or LPS (lipopolysaccharides fromEscherichia coli) as ligands. The capacity of chemokine release was determined by immunoassays.Results Pam3CSK4 and LPS (in combination with thrombin) were able to induce aggregation in WP, but not in PRP, with threshold concentrations of 15 µg/mL. Basal expression levels of TLR2 and TLR4 were higher in WP than in PRP, increasing several-fold rapidly and persistently upon platelet activation with potent agonists. Pam3CSK4 (15 µg/mL) or LPS led to the submaximal release of RANTES, PF4, PDGF, NAP-2, and sCD40L from WP. In PRP, secretory effects are less pronounced for RANTES, PDGF, or PF4, and not detectable for NAP-2 or sCD40L.Conclusion The effects mediated by TLR2 and TLR4 stimulation are dependent on platelet preparation, an important issue for experimental designs and for manufacturing of platelet concentrates in transfusion medicine. Platelets are small nonnucleated cells that are involved in the processes of hemostasis and thrombosis. However, there is growing evidence that platelets play an essential role in mechanisms of inflammation and in immunological reactions.Circulating platelets are among the first cells being confronted with foreign particles at the site of injuries. Platelets themselves have the ability to migrate into tissues and to interact with lymphocytes, dendritic cells, or macrophages modulating their function.TLR2 is able to recognize components of gram-positive bacteria and to exert effects on platelet signal transduction, aggregation, adhesion, or on platelet–neutrophil interactions.In addition, results of previous studies addressing the role of TLR2 and TLR4 for the initiation of platelet aggregation were conflicting.regulated onactivation,normalTcellexpressed andsecreted), or PF4 (platelet factor 4) in PRP.Moreover, the stimulation of TLR2 and TLR4 with bacterial ligands has been shown to result in the activation of platelet signaling cascades accompanied by the shedding of contents.This study, therefore, compared TLR2- and TLR4-dependent platelet responsiveness in PRP and in WP, as commonly used preparation methods for platelet research.Escherichia coliO111:B4 , thrombin, fibrinogen (from human plasma), fetal bovine serum, Tyrode's salt solution, mouse IgG2a (isotype), FITC-conjugated goat antimouse polyclonal antibody, and NAP-2 ELISA Kit were from Sigma-Aldrich Chemie GmbH . Mouse monoclonal anti-TLR2 Adenosine diphosphate (ADP) and thrombin receptor activating peptide-6 (TRAP-6) were from Haemochrom Diagnostica GmbH . Convulxin was obtained from Enzo Life Sciences GmbH , and Pam3CSK4  from InvivoGen . Ethylene glycol-bis(β-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA), prostaglandin E1 (PGE1), epinephrine, LPS fromOur studies with human platelets and the consent procedure were approved by the local ethics committee of the University of Würzburg . All participants provided their written informed consent. The study was performed according to our institutional guidelines and to the declaration of Helsinki.gfor 5 minutes. For the preparation of WP, 3 mM EGTA was added to whole blood to prevent platelet activation. Only the upper 75% of the PRP supernatant was used for experiments to minimize leukocyte contamination with leukocyte concentrations below the detection limit of 0.1 × 103/µL in the blood cell count.Peripheral blood samples from informed healthy voluntary donors  were collected in polystyrene tubes containing 3.2% citrate buffer . PRP was obtained by centrifugation of blood samples at 280 × gfor 10 minutes. The pelleted platelets were washed once in CGS buffer  containing 75 nM PGE1, and resuspended in Tyrode's salt solution.After that, 75 nM PGE1 was added to PRP, and platelets were pelleted at 430 × 8/mL. Blood cell counts were measured with the hematology analyzer KX21N from Sysmex GmbH .Platelet concentrations in PRP and WP were equally adjusted to platelet concentrations of 3.5 × 102was stimulated with Pam3CSK4 diluted in PBS with 0.01% FCS or with LPS. Aggregation was measured for 5 minutes under continuous stirring at 1,000 rpm and 37°C.Light transmission aggregation was measured using an APACT 4004 aggregometer ; 200 µL of PRP or WP suspension supplemented with 1 mM CaClIn LPS studies, samples were costimulated with threshold concentrations  of 0.025 U/mL thrombin for WP and of 1 µM TRAP-6 for PRP. Since the “natural” agonist thrombin is not appropriate for aggregation and flow cytometry studies in PRP due to fibrinogen cleavage and clot formation, TRAP-6 was used.Stimulations with 10 µM TRAP-6 or 0.5 U/mL thrombin served as controls inducing stable aggregation.2was added to platelet suspensions shortly before stimulation. After that, 40 µL of WP was stained with 3 µL of mouse anti-TLR2 or anti-TLR4 antibodies or isotype control for 10 minutes at 37°C. The samples (PRP or WP) were stimulated for 2 to 30 minutes at 37°C with buffer (control), 10 µM TRAP-6, 10 µg/mL convulxin, 10 µM ADP, or with 8 µM epinephrine. Samples were stopped with 1% formaldehyde , fixed for 10 minutes at room temperature, and centrifuged for 1 minute at 14,000 × g. The pellet was resuspended in 100 µL of PBS/BSA/Glc  and stained at room temperature in the dark for 30 minutes with 1 µM of FITC-conjugated goat antimouse antibody. Then, samples were diluted with 300 µL of PBS/BSA/Glc and analyzed by flow cytometry with the FACSCalibur flow cytometer from Becton Dickinson  using CELLQuest software, version 6.0. The platelet population was identified by its forward and side scatter distribution and20,000 events were analyzed for mean fluorescence.For the measurement of TLR2 and TLR4 expression, 40 µL of PRP was stained with 3 µL of anti-TLR2 or anti-TLR4 antibodies or isotype control for 10 minutes at 37°C. For WP, 1 mM CaCl2was added to platelet suspensions before stimulation. After that, samples were stimulated with buffer  or 10 µM TRAP-6 for 2 minutes at 37°C, followed by fixation with 1% formaldehyde  for 10 minutes at room temperature in the dark. Then, samples were diluted with 500 µL of PBS/BSA/Glc and analyzed by flow cytometry as described earlier.For the measurement of CD62P expression, 30 µL of PRP or WP was stained with 3 µL of FITC-conjugated mouse anti-CD62P antibody or isotype control for 10 minutes at 37°C. For WP, 1 mM CaCl2was added to platelet suspensions before stimulation. After that, samples were stimulated with buffer  or 10 µM TRAP-6 for 2 minutes at 37°C, followed by fixation with 1% formaldehyde  for 10 minutes at room temperature in the dark. Then, samples were diluted with 500 µL of PBS/BSA/Glc and analyzed by flow cytometry as described earlier.For the measurement of fibrinogen binding, WP was supplemented with fibrinogen . Then 15 µL of PRP or of fibrinogen-supplemented WP was stained with 15 µL of FITC-conjugated mouse antifibrinogen antibody or isotype control for 10 minutes at 37°C. For WP, 1 mM CaCl2was stimulated with buffer (control), 15 µg/mL Pam3CSK4 diluted in PBS with 0.01% FCS, 15 µg/mL LPS, 10 µM TRAP-6, or 0.5 U/mL thrombin (positive control) for 30 minutes at room temperature. After that, samples were centrifuged for 1 minute at 14,000 × gand supernatants were divided into five tubes and stored at −80°C. The concentrations of released RANTES, PF4, sCD40L, PDGF, and NAP-2 were measured by corresponding immunoassay kits.For the measurement of chemokine levels, 220 µL of PRP or WP suspension supplemented with 1 mM CaCln-values refer to the number of experiments, each made with different blood donors. Data were analyzed by ANOVA using GraphPad PRISM 7  and MedCalc statistic program .p < 0.05 was considered statistically significant.Data are presented as mean ± standard error of the mean (SEM). TheE. coli, as TLR4 agonist.The role of TLR2 and TLR4 for the activation of platelets was analyzed by transmission light aggregometry  using tStimulation of PRP with Pam3CSK4 up to 100 µg/mL did not induce aggregation , whereaTLR4 stimulation with LPS up to 100 µg/mL did not induce aggregation in PRP . SimilaThe expression of TLR2 and TLR4 on the platelet surface was investigated by flow cytometry using receptor-specific antibodies. The extent of induced expression was analyzed with various platelet activators .The basal receptor levels were higher on WP than in PRP. A selective and potent agonist of the platelet collagen receptor glycoprotein VI, 10 µg/mL convulxin increased the expression levels several fold for both receptors in PRP and on WP . SimilaADP (10 µM) as weak agonist stimulated TLR2 and TLR4 expression in PRP approximately by twofold  and lesSince TRAP-6 induced effects were significant for TLR2 and TLR4 in both milieus, we selected TRAP-6 for kinetic studies . Basal TLR2 expression increased to its maximum within 2 minutes after TRAP-6 stimulation on WP and within 5 minutes in PRP. Once activated, TLR2 expression in PRP remained unchanged for the next 30 minutes, whereas it declined on WP within the next 10 minutes, similar to PRP . The peUpon activation, platelets are able to shed chemokines from the α-granules like sCD40L,The concentrations of these chemokines were measured in PRP and in the medium of WP after incubation for 30 minutes with 15 µg/mL Pam3CSK4 or with 15 µg/mL LPS , as thrUpon stimulation with Pam3CSK4 or LPS, the levels of RANTES, PDGF, and PF4 rose significantly, more emphasized in WP compared with PRP .In PRP, neither Pam3CSK4 nor LPS induced higher levels for NAP-2 or sCD40L . HoweveCompared with stimulation with 15 µg/mL Pam3CSK4 or LPS, the chemokine concentrations reached much higher levels upon strong platelet activation with TRAP-6 or thrombin . The vaThe activation levels of WP and PRP were measured by flow cytometric analysis of CD62P expression  and fibThere are several studies dedicated to TLR expression and function in platelets. However, many results are hardly comparable since some of the studies investigated WP,According to previous studies,The elevation of receptors, in WP and PRP, was more pronounced with potent platelet agonists like convulxin and TRAP-6 than with weak agonists like ADP or epinephrine. This upregulation of TLR2 and TLR4 expression is similar to other ligands or receptors , which are recruited from platelet α-granules to the platelet surface upon agonist stimulation. The washing procedure contributes to slight preactivation of platelets, indicated by higher levels of CD62P and fibrinogen binding in comparison to PRP, and in that way, presumably contributing to higher TLR2 and TLR4 expression levels in WP. According to our results, an increase in TLR4 levels was reported in thrombin-stimulated WP by Tsai et al,In general, it is well known that agonist-stimulated platelets release several immunomodulating ligands and cytokines like sCD40L, PF4, RANTES, PDGF, or NAP-2.As the lowest concentrations promoting platelet aggregation, 15 µg/mL Pam3CSK4 and 15 µg/mL LPS were able to induce the shedding of all investigated chemokines from WP (approximately two- to fivefold compared with unstimulated controls), supporting the results of aggregation studies with the potential of TLR2 and TLR4 for platelet activation. In this context, the capacity of TLR2 and TLR4 to mediate the release of NAP-2 has been shown for the first time. Compared with strong agonists like thrombin or TRAP-6, the secretory effects were submaximal and more pronounced with the TLR2 ligand Pam3CSK4. In contrast, in PRP, the induced increment was smaller (∼1.3- to 2-fold), the peak concentrations of chemokines were lower than in WP, and there was no increase of NAP-2 and sCD40L. Such a remarkable difference for sCD40L was observable even after maximal platelet activation with TRAP-6 or thrombin.In consequence, plasma factors should be considered as relevant modulators of chemokine levels  were used for stimulation, but future studies should confirm the findings by additional TLR2 and TLR4 ligands. Obviously, TLR2 exerts its effects via the phosphoinositide 3-kinase (PI3-K)/Akt pathwayAs a limitation of the study, it must be considered that expression patterns and secretory responses may be influenced variably by different procedures for washing platelets or by different storage media. However, methods and conditions in this study have been commonly used for experimental platelet research.This direct comparison of WP and PRP has rendered several novel contributions to the understanding of TLR2- and TLR4-mediated platelet responsiveness. The dependence of platelet preparation on TLR integrity is an important issue for the design of experimental settings in basic research. In addition, the results are of clinical relevance . Plasma reduction is discussed to prevent adverse transfusion reactions by removing accumulated cytokines.In summary, platelet activation results in a rapid and sustained increase of TLR2 and TLR4 expression in both milieus. In WP, but obviously not in PRP, receptor stimulation can induce (TLR2) or support (TLR4) platelet aggregation. In WP, TLR2 and TLR4 stimulation promotes the subtotal release of chemokines from α-granules, shown for NAP-2 for the first time. In PRP, the secretory effects are less pronounced for RANTES, PDGF, or PF4, and not detectable for NAP-2 or sCD40L.


Arabidopsis SWR1-C has not been thoroughly characterized. Here, we aim to identify interacting partners of a conserved Arabidopsis SWR1 subunit ACTIN-RELATED PROTEIN 6 (ARP6). We isolate nine predicted components and identify additional interactors implicated in histone acetylation and chromatin biology. One of the interacting partners, methyl-CpG-binding domain 9 (MBD9), also strongly interacts with the Imitation SWItch (ISWI) chromatin remodeling complex. MBD9 is required for deposition of H2A.Z at a distinct subset of ARP6-dependent loci. MBD9 is preferentially bound to nucleosome-depleted regions at the 5’ ends of genes containing high levels of activating histone marks. These data suggest that MBD9 is a SWR1-C interacting protein required for H2A.Z deposition at a subset of actively transcribing genes.Deposition of the histone variant H2A.Z by the SWI2/SNF2-Related 1 chromatin remodeling complex (SWR1-C) is important for gene regulation in eukaryotes, but the composition of the  Arabidopsis.The SWI2/SNF2-Related 1 chromatin remodeling complex (SWR1-C) is important for gene regulation, but its composition remains largely uncharacterized in plants. Here, the authors report that methyl-CpG-binding domain 9 (MBD9) is a SWR1-C interacting protein required for histone H2A.Z deposition in  Cells contain chromatin remodeling complexes, which modulate the packaging, spacing, and composition of nucleosomes3. The activity, targeting, and composition of remodeling complexes is tightly regulated to enable proper development of an organism3.Chromatin dynamics plays a central role in regulating gene expression and has important implications in various cellular processes. The basic unit of chromatin is the nucleosome, which consists of 147 base-pairs (bp) of DNA wrapped around a histone octamer composed of two of each of the histones H2A, H2B, H3, and H4 , with a particular preference for the +1 nucleosome10. The relationship between H2A.Z accumulation and transcription is complex. When present in the active genes of some organisms, the levels of H2A.Z at the TSS positively correlate with gene expression, typically when found together with H3/H4 acetylation, marks of active transcription13. H2A.Z is nonetheless absent at the most highly expressed genes7 and is also localized around the TSS of repressed genes that are poised for activation in yeast and animals16. Although the function of gene body methylation is unclear, one study in Arabidopsis suggests that a function of gene body DNA methylation may be to prevent H2A.Z deposition over housekeeping genes17, while another study found no such evidence18. However, it is clear that an anti-correlation between H2A.Z and DNA methylation exists across diverse systems21. In Arabidopsis, genes bearing high levels of gene body DNA methylation tend to be long and highly expressed, and show H2A.Z enrichment at their 5′-ends overlapping with activating histone modifications24. By contrast, enrichment of H2A.Z over gene bodies (including high H2A.Z levels at the TSS) is typically found in shorter, weakly expressed genes involved in environmental/stress responses that are marked with H3K27me3 (refs. 25). Recently, levels of H3K27me3 were shown to be dependent on H2A.Z deposition in Arabidopsis26. Together, these studies demonstrate that H2A.Z can participate in both activation and repression of gene expression, likely in concert with other factors and chromatin marks.The most highly conserved histone variant in eukaryotes is H2A.Z29. In yeast, the SWR1 complex (SWR1-C) is composed of 14 subunits30. In mammals, the structurally and functionally equivalent homolog of the yeast SWR1 complex, Snf2-Related CREBBP Activator Protein (SRCAP), is composed of 11 subunits, which are also conserved in yeast33. Based on homology, Arabidopsis contains one or several orthologs of the 11 conserved subunits from the yeast and human SWR1/SRCAP complexes, respectively34. Based on genetic and protein–protein interaction studies, several components have been validated as components of the Arabidopsis SWR1 complex, including PHOTOPERIOD-INDEPENDENT EARLY FLOWERING 1 (PIE1), the ATPase/scaffold subunit; ACTIN-RELATED PROTEIN 6 (ARP6); SERRATED LEAVES AND EARLY FLOWERING (SEF), the homolog of the yeast SWC6; and SWR1 complex subunit 2 (SWC2)37. A recent pull-down study of the SWC6 subunit identified additional subunits of the Arabidopsis SWR1 complex in yeast and validated their interaction with SWR1 complex subunit 4 (SWC4)38. Mutations in the genes encoding the SWR1 subunits share various phenotypes, with null mutations in the core catalytic subunit PIE1 producing the most severe phenotypes, including reduced size, loss of apical dominance, reduced fertility, and early flowering41.H2A.Z is deposited by the SWI2/SNF2-Related 1 (SWR1) complex, which involves the replacement of H2A-H2B dimers by H2A.Z-H2B dimers46. The SWR1-C and NuA4 complexes share four common members: SWC4, YEAST ALL1-FUSED GENE FROM CHROMOSOME 9 (YAF9), ACTIN-RELATED PROTEIN 4 (ARP4), and ACTIN 1 (ACT1)49. Homologs of these four proteins are present in the mammalian TIP60 complex, which possesses the dual activities of acetylating histones and depositing H2A.Z51. The Arabidopsis genome also contains homologs for many of the yeast NuA4 subunits; however, a functional link between histone acetylation and H2A.Z deposition is unclear52. YAF9 proteins are required for both the proper expression of FLOWERING LOCUS C (FLC) and H2A.Z acetylation at its regulatory regions, but not for H2A.Z deposition53. Much more needs to be learned about the potential NuA4 complex in Arabidopsis, including its composition and genome-wide effects on H2A.Z deposition and modification. Although genome-wide localization of the SWR1 components is lacking in Arabidopsis, it has been reported that ARP6 and SWC4 directly bind their target locus FLOWERING LOCUS T (FT)38; likewise, ARP6 and SWC6 bind directly to FLC35.How the SWR1 complex is targeted across the genome remains unclear. Evidence suggests that H2A.Z localization around TSS in yeast is dependent on open chromatin and histone acetylation deposited by the Nucleosome acetyltransferase of H4 (NuA4) complexArabidopsis SWR1 complex. We identify Arabidopsis homologs of most of the conserved SWR1 complex components in yeast, as well as several additional interacting partners: MBD9, four of the seven members of the ALFIN1-LIKE family , and two components of the yeast NuA4 and Arabidopsis SAGA histone acetyltransferase complexes, TRANSCRIPTION-ASSOCIATED PROTEIN 1A AND 1B (TRA1A and TRA1B). Our data are consistent with a related study where ARP6-interacting proteins were identified using ARP6-TAP-tag purification followed by mass spectrometry54. We focus on characterizing MBD9, a protein implicated in flowering time regulation55. Using biochemical, genetic, and transcriptome analyses, we show that MBD9 interacts with the SWR1 complex in Arabidopsis, and is required for H2A.Z deposition at a subset of ARP6-dependent genes. Furthermore, immunoprecipitation of MBD9 followed by mass spectrometry (IP–mass spec) reveals its association with the ISWI chromatin remodeling complex, suggesting additional functions for MBD9. Genome-wide ChIP-seq analyses of MBD9 show that it localizes to nucleosome-depleted regions of active genes but does not influence open chromatin itself. MBD9 is thus an interacting partner of the Arabidopsis SWR1 complex required for H2A.Z deposition at a subset of actively transcribed genes.In this study, we use the conserved ARP6 subunit as bait to analyze the composition of the Arabidopsis, we used the conserved ARP6 subunit of the SWR1 complex as bait. We generated the epitope-tagged ARP6 lines pARP6:ARP6-3xFLAG and pARP6:ARP6-9xMYC. Both lines complemented arp6-1 mutants , ARP4, SWC4, SWC2, YAF9A, and SEF. This includes three of the four common subunits of the yeast NuA4 complex, ARP4, SWC4, and YAF9A  and AL4, AL5, AL6, and AL7, which recognize di- and trimethyl H3K4 histone marks57. The interaction between ARP6 and MBD9 was further validated by co-immunoprecipitation (co-IP) in Arabidopsis flowers using an antibody raised against MBD9 . Both single mutants displayed an early flowering time phenotype  and short-day (SD) conditions  was slightly shorter under SD growth conditions in the double mutant than the arp6-1 mutant , indicated that both the single mutants and the double mutant had a similar reduction of expression of these genes , we defined common H2A.Z differential regions for each genotype . These genes were enriched for cell cycle progression functions  according to the levels of H2A.Z in wild type over gene bodies  and TSS  as described in ref. 17 , whereas loss of H2A.Z in the arp6-1 mutant tends to occur over genes containing high levels of H2A.Z over gene bodies  , gen9-3 Fig. . We alsoes) Fig. .mbd9-3, arp6-1, and arp6-1 mbd9-3. We defined differentially expressed genes  did not show a significant change between arp6-1 and arp6-1 mbd9-3  and those specifically up-regulated in arp6-1 mbd9-3 (n = 218)  had minimal if any change in H2A.Z levels in mbd9-3  genes misregulated in the mbd9-3 background are a subset of genes misregulated in the arp6-1 background; (3) MBD9 and ARP6 also cooperate to control the expression of a subset of up-regulated genes; and (4) although loss of H2A.Z is observed genome-wide in arp6-1, and is even more pronounced in arp6-1 mbd9-3, genes that change in expression upon the loss of H2A.Z are generally those that contain high levels of H2A.Z at TSS and high-medium levels of H2A.Z over the gene body.We compared differentially expressed genes in relation to H2A.Z changes in the mutants. First, to obtain general trends, we generated boxplots of expression and profiles of H2A.Z in the nine classes of genes divided according to their H2A.Z occupancy , we defined 10,348 MBD9-occupied peaks, corresponding to 5772 genes having a peak within 1 kb upstream of the TSS  to reflect typical patterns of H2A.Z occupancy in Arabidopsis17  and low levels of H3K27me3, and coincided with MBD9-occupied peaks and open chromatin. In agreement, a related study also identified an enrichment of histone acetylation marks at MBD9-dependent H2A.Z sites54.To determine if specific chromatin features mark the regions that are enriched with MBD9, we analyzed ATAC-seq data from wild-type plants and observed that MBD9-occupied genes tended to have more open chromatin than either an equivalent set of genes with similar expression levels, or all protein-coding genes Fig. . We alsoelopment . MBD9-ocelopment . A similelopment , whereaselopment . This geAltogether, these results suggest that MBD9 preferentially localizes to the open chromatin regions of actively transcribing genes, which contain high levels of H3K4me3 and histone acetylation and are different from genes controlled by the Polycomb Group Complex.52. How it is specifically targeted to the locations where these events occur is poorly understood. In this study we have characterized the interacting partners of Arabidopsis ARP6, a homolog of Arp6 found in the yeast SWR1-C involved in H2A.Z deposition. We identified nine Arabidopsis proteins that are homologous to subunits common to the yeast (SWR1) and mammalian (SRCAP) complexes, ARP6, PIE1, RIN1, RIN2, ARP4, SWC4, SWC2, YAF9A, and SEF. We further identified several additional ARP6-interacting partners, TRA1A, TRA1B, MBD9, and four members of the Alfin1-like family . These Alfin1-like-containing proteins recognize H3K4me3 histones57, suggesting that they play a role in recruiting the SWR1 complex to H3K4me3 regions. The ARP6-interacting partners identified in our study are in agreement with a related study involving ARP6-TAP purification followed by mass spectrometry54. However, there were a few differences. Specifically, we did not enrich for H2A.Z or ACT1 in our mass spec data, perhaps reflecting different methods of isolation, while we did identify TRA1A and AL4 as ARP6-interacting partners that were not reported by Sijacic et al.54.H2A.Z has been implicated in a wide variety of functions, including transcription, meiosis, and DNA damage65, MADS proteins66, and DTT-containing proteins67. MBD9 was among the reported interacting partners of CHR17, independently validating our results65. However, only two other SWR1 complex components (ARP4 and RIN1) were detected in the same CHR17 IP–mass spec study. Since we did not detected preferential enrichment between CHR11/17 and ARP6 in our data, this suggests that MBD9 interacts with the two complexes separately. Our analysis of the genetic interactions between mbd9-3 and arp6-1 suggests that, despite the more severe developmental phenotype of the double mutant, ARP6 is epistatic to MBD9 regarding flowering time. These two observations are in agreement since flowering time is controlled by a particular set of genes, whereas H2A.Z deposition is globally affected in our mutants. The stronger developmental phenotype of the double mutant could have two plausible explanations: (1) multiple pathways are involved or (2) since the double mutant closely resembles pie1-2 , the phenotype results from MBD9 and ARP6 being part of the SWR1 complex, where the complex may be partly functional without one of the components but not without both. Future work will discriminate between these possibilities. Our genomic observations, H2A.Z ChIP-seq data, and transcriptome analysis revealed that MBD9 and ARP6 share many common targets, but the mbd9-3 mutant shows a weaker phenotype than arp6-1. We observed that MBD9 is required for the deposition of H2A.Z at a subset of arp6-1 dependent loci, similar to what was reported by Sijacic et al.54. We also identified a subset of genes that require both ARP6 and MBD9 for both their expression and for H2A.Z deposition, suggesting the two proteins act redundantly in some cases. Additionally, we determined the genome-wide occupancy of MBD9 and showed that it preferentially localized to the open chromatin regions of promoters and the transcription termination sites of active genes that have high levels of activating histone modifications (H3K4me3 and histone acetylation) and did not localize to genes repressed by the Polycomb Group Complex. These data suggest that the MBD9-containing SWR1 complex promotes H2A.Z deposition at the 5′ ends of highly expressed genes.We focused on characterizing the interaction between ARP6 and MBD9. We identified interacting partners for MBD9 and showed that it interacted with most components of the SWR1 complex, including PIE1, RIN1, ARP4, RIN2, SWC2, ARP6, SWC4, YAF9A, and SEF. MBD9 also interacted with TRA1A and TRA1B, further implicating histone acetylation in H2A.Z deposition. Additionally, we detected high enrichment of the ISWI chromatin remodeling complex in our MBD9 IP, which was not seen in the ARP6 IP–mass spec. The ISWI remodeling complex interacts with other chromatin remodelersArabidopsis, and (3) MBD9 only transiently interacts with the SWR1 complex. Based on size exclusion chromatography of ARP6-containing SWR1 complex in the mbd9 mutant, Sijacic et al.54 concluded that MBD9 is not a core subunit of the Arabidopsis SWR1 complex. However, as noted by the authors, this does not exclude the possibility that MBD9 is part of a small subset of all SWR1 complexes in Arabidopsis54. Given that we detected most components of the SWR1 complex in our MBD9 IP–mass spec, our genetic and genomic data best fit the model that MBD9 is part of a subset of the SWR1-containing complexes.Regarding the interaction of MBD9 with the SWR1 complex, three possibilities exist based on our genetic and genomic observations: (1) MBD9 is always a member of the SWR1 complex but not always required, (2) MBD9 is part of a subset of all SWR1 complexes in mbd9-3 mutant, this loss did not result in major transcriptional changes. This may be because the loss of H2A.Z in the mbd9-3 mutant occurs over genes with low H2A.Z over gene bodies and several studies suggest that genes changing most upon loss of H2A.Z are the ones bearing H2A.Z over the gene body24. One plausible explanation for the lack of transcriptional response at these regions in the mbd9-3 mutant is redundancy. It is possible that redundant mechanisms are present at housekeeping genes to maintain their high expression and losing H2A.Z is not enough to perturb their activity. In support, H2A.Z is proposed to have redundant roles with chromatin remodeling complexes in yeast68. Interestingly, we identify components of the ISWI chromatin remodeling family as interacting partners of MBD9 in our IP. Additionally, CHR11/17 mutants have defects in flowering time and decreased levels of FLC expression69. Perhaps in addition to promoting H2A.Z deposition, MBD9 promotes low nucleosome occupancy at genes containing H2A.Z at +1 by interacting with chromatin remodelers to maintain these loci in a transcriptionally permissive state. In support, a recent study of FORGETTER1, a protein involved in heat-shock memory response, was found to interact with chromatin remodelers including CHR11/17 to maintain low nucleosome occupancy of genes in non-inductive conditions65. In Arabidopsis, the ISWI remodeling complex is required for the regular spacing of nucleosomes downstream of the +1 border nucleosome70. Interestingly, many highly expressed genes in the CHR11/17 mutant have defects in nucleosome positioning, yet their transcription is unaffected70. This finding supports our hypothesis that redundant mechanisms maintain high expression of housekeeping genes. Higher order mutants between the SWR1 complex and chromatin remodelers are required to test this hypothesis.Surprisingly, although we detected many genes that lost H2A.Z in the 43. Consistent with the role of MBD9 in the SWR1 complex, we found that MBD9 also localizes to nucleosome-depleted regions, in close proximity to H2A.Z deposited at the 5′-ends of gene bodies. These regions are devoid of DNA methylation, consistent with the prediction that the methyl-binding domain of MBD9 does not recognize methylated sequences61.In yeast, the SWR1 complex preferentially binds to nucleosome-depleted regions50. In Arabidopsis, TRA1B interacts with SWC4, a component of the predicted NuA4 complex71, and both TRA1A and TRA1B are components of the Arabidopsis SAGA acetyltransferase complex72. This suggests that various acetyltransferase complexes may be promoting H2A.Z deposition via histone acetylation. Since we did not detected the NuA4 scaffold or the catalytic subunit in our IP, it is unlikely that the ARP6-containing SWR1 complex represents a fusion of the Arabidopsis SWR1 and NuA4 complexes, similar to the mammalian TIP60 complex; rather, the TRA1A and TRA1B subunits may function to bridge the acetyltransferases with the SWR1 complex.The genes bearing MBD9 also contain high levels of histone acetylation, consistent with the observation that histone acetylation in yeast promotes H2A.Z deposition. We also observed enrichment of three of the four common NuA4 histone acetylation complex subunits, SWC4, YAF9, and ARP4, in our IP–mass spec. We also identified two homologs of the yeast Tra1 proteins, TRA1A and TRA1B, in both ARP6 and MBD9 IPs. Tra1 is a component of the yeast NuA4 complexArabidopsis and shed light on the role of MBD9 in promoting H2A.Z deposition at a subset of highly expressed genes. Further work addressing the function of the H2A.Z histone variant should reveal the nature of its dependency on factors such as histone acetyltransferase and chromatin remodeling complexes for carrying out its varied functions across the genome.In summary, our study identified components of the SWR1 complex in Arabidopsis plants used in this study were of the Col-0 ecotype and were grown at 22 °C under long-day conditions . For the short-day flowering time experiment, plants were grown at 22 °C . The following Arabidopsis mutant lines were used: mbd9-3 (SALK_039302), arp6-1 (Sail_599_G03), and pie1-2 (SALK_003776C).All Total RNA was prepared from whole seedlings (14 DAG) using the RNA Prep Pure Plant Kit . Two micrograms of RNA was used for the preparation of cDNA using PrimeScript Reagent Kit with gDNA eraser . Signal detection, quantification, and normalization were done using Quant Studio 6 proprietary Software .−1 were used per sample. Approximately 2 g of floral tissue was ground in liquid nitrogen, 10 mL IP buffer ) was added, the mix was incubated on ice for 20 min, and spun at 4000g for 10 min at 4 °C. The supernatant was filtered through a double layer of Miracloth. Anti-MBD9 antibody coupled to magnetic beads was added and incubated for 2 h at 4 °C with gentle rotation. Beads were washed six times with IP buffer, 5 min each wash at 4 °C with rotation, and then boiled. Western blotting and detection were performed with anti-Myc 9E10 mouse monoclonal antibody  with 1:1000 dilution.An anti-MBD9 polyclonal antibody was raised in rabbits using the 1941–2176 aa MBD9 peptide as an antigen. The antibody was coupled to magnetic beads using the Dynabeads antibody coupling kit  following the manufacturer’s recommendations; 7 µL of 3.12 mg mLTotal RNA was extracted from 13 DAG shoots grown on 1% MS supplemented with 1% sucrose under long-day conditions. Four replicates, grown on separate plates, were collected for each genotype. RNA was extracted using the Direct-zol RNA Miniprep kit (Zymo). For RNA-seq, 1 µg of total RNA was used to prepare libraries using the TruSeq Stranded mRNA-Seq kit (Illumina). Reads were aligned to TAIR10 using Tophat by allowing up to two mismatches and mapping only to one location. FPKM values and differential gene expression were analyzed using Cufflinks with default settings. GO term enrichment was determined using AgriGO.Full-length genomic DNA fragments containing ~1 kb (MBD9) or ~1.5 kb (ARP6) of promoter sequence, together with genomic gene sequences up to the annotated/major stop codon of MBD9 and ARP6 were amplified using PCR (primers are listed in Supplementary Table −1) and suspended in 30 mL of IP buffer. Tissue was further dounce homogenized until lump-free, then centrifuged for 20 min at 4000g and 4 °C. The lysate was filtered through two layers of Miracloth. Supernatant was incubated with 200 μL anti-FLAG M2 magnetic beads , at 4 °C for 2 h. The bead-bound complex was washed once with 10 mL IP buffer, then four times (5 min rotating at 4 °C with 1.5 mL IP buffer), followed by a final wash with IP buffer without NP-40. The FLAG-IP was eluted twice with 300 µL 250 µg mL−1 3X FLAG peptides  in TBS , mixing for 15 min at 4 °C. The eluted protein complexes were precipitated by trichloroacetic acid and subjected to mass spectrometric analyses. In the case of ARP6-9xMyc and MBD9-9xMyc, monoclonal 9E10 coupled to magnetic beads was used . The bead-bound complexes were washed six times with 1 mL of IP buffer. For each wash, the beads were rotated at 4 °C for 5 min. For ARP6-9xMyc, proteins were released from the Streptavidin beads by 3C cleavage overnight at 5 °C. 3C-GST was removed by incubation with GST couple magnetic beads . For MBD9-9xMyc, proteins were eluted twice with 100 µL 8 M Urea in 50 mM Tris pH 8.5, mixing for 15 min at 37 °C. The supernatant was TCA precipitated.Approximately 10 g of flowers from transgenic lines expressing T3 ARP6-3xFLAG or T4 MBD9-3xFLAG and from Col-0 plants as negative controls were ground to a fine powder using a RETCH homogenizer  antibody was used for H3 ChIP assays (10 µL of 0.5 mg mL−1) and active motif 39140 (Active Motif) was used for pan-acetyl H3 ChIP assays (5 µL of 1 mg mL−1).For histone modification ChIPs, about 0.5 g of shoots were collected from 13 DAG seedlings grown on 1% MS plates supplemented with 1% sucrose under long-day conditions. Samples were crosslinked in vitro, sheared using Bioruptor Plus (Diagenode). Libraries were generated with NuGEN Ovation Ultra Low System V2 kit, according to the manufacturer’s instructions, and were sequenced on an Illumina HiSeq 4000 instrument. H2A.Z ChIP assays were performed using polyclonal antibodies specific to −1) and resuspended with Nuclear Isolation Buffer . Lysate was filtered through one layer of Miracloth and centrifuged for 20 min at 2880g and 4 °C. The pellet was resuspended with 1 mL Extraction buffer 2 ), followed by centrifugation for 10 min at 12,000g and 4 °C. The pellet was then resuspended in 500 µL Extraction buffer 3 , layered over 500 µL extraction buffer 3 and centrifuged for 1 h at 12,000g and 4 °C. The pellet was lysed with 400 µL Nuclei Lysis Buffer on ice . To lysed nuclei, 1.7 mL of ChIP Dilution Buffer  was added and DNA was sheared on a Bioruptor Plus (Diagenode) . Sheared chromatin was centrifuged twice at max speed for 10 min at 4 °C. The supernatant was further diluted with ChIP dilution buffer up to 4 mL. One hundred microliters of sample were saved as input and the rest was split into 2 mL DNA LoBind tubes  and 10 µL of 1 mg mL−1 anti-Flag antibody was added . After incubation overnight with rotation at 4 °C, 50 µL Dynabeads  was added to chromatin and incubated for an additional 2 h. The magnetic beads were washed with 1 mL of the following buffers for 5 min rotating at 4 °C: 2× with low salt buffer , 1× high salt buffer , 1× LiCl Wash Buffer , and 1× with TE buffer . The immunocomplex was eluted from the beads twice with 250 µL elution buffer , incubating for 20 min with shaking at 65 °C. Four hundred microliters of elution buffer was added to the input samples. A total of 20 µL 5 M NaCl was added to each tube; the crosslink was reversed by incubation at 65 °C overnight. Residual protein was degraded with 20 µg Prot K in 10 mM EDTA and 40 mM Tris pH 8 at 45 °C for 1 h followed by phenol/chloroform/isoamyl alcohol extraction and ethanol precipitation. The pellet was washed with 70% EtOH and resuspended in 50 µL molecular grade water. Libraries were prepared using the NuGEN Ovation Ultra Low System V2 kit, according to the manufacturer’s instructions, and were sequenced on an Illumina HiSeq 4000 instrument.For MBD9, about 5 g of 13 DAG T4 MBD9-3XFLAG and Col-0 seedlings grown on 1% MS were fixed in vivo using vacuum infiltration as follows. Protein–protein crosslinking was performed first using 1.5 mM EGS in 1× phosphate-buffered saline (PBS) for 10 min followed by three washes with PBS. Next, tissue was fixed with 1% formaldehyde in 1× PBS for 20 min. Crosslinking was stopped with 0.125 M glycine in 1× PBS and the seedlings were vacuum infiltrated for 5 min. Tissue was patted dry and frozen at −80 °C. Tissue was homogenized to a fine powder using a RETCH homogenizer (2 min @ 30 Hz mink-means clustering were generated by NGSPLOT (v4.02.48). Enriched motifs in peak sets were identified using HOMER findMotifsGenome. Annotation of peak locations were carried out using the HOMER annotatepeaks function. GO term enrichment was determined using AgriGO.ChIP-seq fastq reads were aligned to the TAIR10 reference genome with Bowtie using default settings and allowing only uniquely mapping reads. Duplicated reads were removed using SAMtools. IGB genome browser was used to visualize the data and to generate snapshots. Normalized read coverage tracks were generated using the USeq package Sam2Useq application. ChIP-seq peaks in wild type and mutants were called by callpeak function in MACS2 (v2.1.1.). ChIP-seq data metaplots and 2, 5 mM KCl, 0.2% Triton X-100, 5 mM β-mercaptoethanol, 35% glycerol). The samples were ground with an Omni International General Laboratory Homogenizer at 4 °C and then filtered through a two-layer Miracloth and a 40-µm nylon mesh Cell Strainer (Fisher). Samples were spin filtered for 10 min at 3000g, the supernatant discarded, and the pellet resuspended with 25 mL of grinding buffer using a Dounce homogenizer. The wash step was repeated once and the nuclei resuspended in 0.5 mL of freezing buffer . Nuclei were subjected to transposition reaction with Tn5 73. For transposition reaction, 25 µL of 2× DMF (66 mM Tris-acetate (pH = 7.8), 132 K-Acetate, 20 mM Mg-Acetate, 32% DMF) was mixed with 2.5 µL Tn5 and 22.5 µL nuclei suspension and incubated at 37 °C for 30 min73. Transposed DNA fragments were purified with ChIP DNA Clean & Concentrator Kit 73. Libraries were generated using Phusion High-Fidelity DNA Polymerase  as follows, 12.5 µL 2× Phusion, 1.25 µL 10 mM Ad1 primer, 1.25 µL 10 mM Ad2 primer, 4 µL ddH2O and 6 µL purified transposed DNA fragments were mixed and subjected to 12 cycles of PCR amplification 73. Libraries were sequenced on an Illumina HiSeq 2000 instrument. ATAC-seq read adaptors were removed with trim_galore and then mapped to the Arabidopsis thaliana reference genome TAIR10 using Bowtie (-X 2000 -m 1). Chloroplast and mitochondrial DNA aligned reads were filtered out and duplicate reads were removed using SAMtools.Inflorescence tissues were collected for nuclei extraction as follows. About 5 g of inflorescence tissue was collected and immediately transferred into ice-cold grinding buffer (300 mM sucrose, 20 mM Tris pH 8, 5 mM MgClFurther information on research design is available in the Supplementary InformationReporting Summary


Tg) of PHAs is discussed. The introduction of aromatic monomers into PHA chains is a promising method for improving the properties of PHAs, as the characteristics of aromatic PHAs differ from those of aliphatic PHAs.Polyhydroxyalkanoates (PHAs) are polyesters synthesized by bacteria as a carbon and energy storage material. PHAs are characterized by thermoplasticity, biodegradability, and biocompatibility, and thus have attracted considerable attention for use in medical, agricultural, and marine applications. The properties of PHAs depend on the monomer composition and many types of PHA monomers have been reported. This review focuses on biosynthesized PHAs bearing aromatic groups as side chains. Aromatic PHAs show characteristics different from those of aliphatic PHAs. This review summarizes the types of aromatic PHAs and their characteristics, including their thermal and mechanical properties and degradation behavior. Furthermore, the effect of the introduction of an aromatic monomer on the glass transition temperature ( Ralstonia eutropha and Pseudomonas putida. PHAs are accumulated as an intercellular energy and carbon storage material under nutrient-limited conditions in the presence of excess carbon sources  has a Tm of 138–147 °C and Tg of 0 °C , and P(3-hydroxy-5-(para-fluorophenoxy)valerate-8.7 mol % 3-hydroxy-7-(para-fluorophenoxy)heptanoate) [P(3H5pFPxV-8.7 mol % 3H7pFPxHp)] were produced from 11-(meta-fluorophenoxy)undecanoic acid and 11-(para-fluorophenoxy)undecanoic acid, respectively. When 11-undecanoic acid was used as the carbon source, the Pvalerate) [P(3H5opFPxV)] homopolymer was produced. These PHAs showed water-shedding properties and higher thermostability than other aromatic PHAs  [P(3HA-3HPhA)] became glue-like as the acyl chain length of the supplied ω-phenylalkanoic acid was increased  with various 3H5PhV contents is summarized in The surface properties of P(3H5opFPxV) with two fluorine atoms were evaluated . The surThe degradability of aromatic PHAs has also been studied. Degradability is one important property for the use of PHAs as biodegradable materials. For medical applications such as drug delivery systems, the stability of PHAs at physiological pH and the safety of the material released through hydrolysis should be evaluated by evaluating the chemical degradation. For agricultural and marine applications, degradation by microorganisms should also be studied.β-oxidized in vivo to phenylbutyric acid, phenylacetic acid, or trans-cinnamic acid, may exert important pharmaceutical effects, thereby improving or broadening the clinical effects of the encapsulated drug. Olivera et al. synthesized polymeric microspheres of P(3H6PhHx) at atmospheric pressure by a solvent evaporation method, and demonstrated its potential use as a drug vehicle [The chemical degradation of the P(3H6PhHx) homopolymer was analyzed, as reported in the literature . This po vehicle .R)-3-hydroxy-ω-phenylalkanoates (C5–C8), a hydrolytic product of PHAs bearing a phenyl group, is established [R)-3-hydroxy-ω-phenylalkanoates inhibited the growth of Listeria species, attributed only (or mainly) to the (R)-enantiomer.The antibacterial activity of , which have Tgs in the range of −53 to −28 °C [Tg resulted from the introduction of aromatic monomers have been also reported in other aromatic monomers as described below. This behavior is different from mcl-PHAs. On the other hand, the Tm of P(3H5PhV) ranged from 54 to 69 °C, which is similar to those of the n-alkyl-substituted PHAs. As for the value of Tm, P(3H5PhV) is most similar to mcl-PHAs. However, the endothermic peak for the melting transition in the differential scanning calorimetry (DSC) thermogram was very small. This observation indicates that this polymer has a very low degree of crystallinity and is thus highly amorphous [n-alkyl-substituted PHAs [P(3H5PhV), the first biosynthesized PHA bearing an aromatic group, has a of 13 °C . This ismorphous ,27,52. Tmorphous . P [Tg may reflect decreased intermolecular interaction between the backbone chains, thereby resulting in increased backbone chain mobility, caused by lengthening of the side-chains by one methylene unit relative to that of 3H5PhV [The P(3H4PhB-95 mol % 3H6PhHx) copolymer has a (3H5PhV) . Similar(3H5PhV) . The lowf 3H5PhV . This saf 3H5PhV ,19. ThisTg than that of mcl-PHA [Tg values followed the order: P(3H5PhV-38 mol % 3H6PhHx-50 mol % 3H7PhHp) > P(3H5PhV-77 mol% 3H7PhHp) > P(3H6PhHx-73 mol % 3H8PhO). This is attributed to the increasing flexibility of the PHA chains due to the introduction of structural irregularities and alkyl side chains longer than three methylene units.Similarly, other PHAs composed of only monomers bearing phenyl groups produced from phenylalkanoic acids showed the same characteristics, being totally amorphous and with a higher  mcl-PHA ,40. In t mcl-PHA , the Tg Tg at −20 °C [Tgs at −31 and 5 °C [Tg of the polymer produced from NA alone and P(3H5PhV), this sample is deduced to be a mixture of two PHAs. As described in Tgs.PHAs consisting of 3HA and 45 mol % 3H5PhV as aliphatic and aromatic monomers, exhibited only one t −20 °C . On the and 5 °C . BecauseP. putida produced P(3HA-98 mol % 3H5PhV) and P(3HA-15 mol % 3H4PhB-83 mol % 3H6PhHx) from 5PhV and 6PhHx, respectively, where the PHAs were partially crystalline with Tms of 51.5 and 52.1 °C, respectively [Tm, indicating that these polymers were totally amorphous. The Tg of these polymers varied between 13.2 °C for P(3HA-98 mol % 3H5PhV) accumulated from 5PhV to −14.3 °C for P(3HA-7 mol % 3H4PhB-61 mol % 3H6PhHx-30 mol % 3H8PhO) accumulated from 8PhO. The decomposition temperature decreased as the length of the acyl side chain of the aromatic substrate was increased (283 °C for P(3HA-98 mol % 3H5PhV) produced from 5PhV to 254 °C for P(3HA-6 mol % 3H4PhB-57 mol % 3H6PhHx-26 mol % 3H8PhO-9 mol % 3H10PhD) from 10PhD) [The effect of incorporation of the phenyl monomer into P(3HA) on the thermal properties has been studied, and the effects of the types of aromatic monomers were investigated . P. putiectively . Howeverm 10PhD) .Tg increased from −49.3 to 5.90 °C and the Tm decreased from 82.4 to 50.40 °C. Therefore, the thermal properties of the P(3HA-3HPhA) copolyester can be easily modified by changing the monomer content, which leads thermal properties intermediate between those of the two homopolymers.The effects of the aromatic monomer ratio were also investigated . With anω-alkenoates(3HE)) was analyzed by Hartmann and coworkers [Tg increased linearly from −38.7 to −6.0 °C as the 3H5PhV content increased from 0 mol % (P(3HA-10 mol % 3HE)) to 59 mol % (P(3HA-10 mol % 3HE-59 mol % 3H5PhV)). The estimated Tg of P(3H5PhV) calculated from these values is about 14.1 °C, and is in good agreement with the literature value (13 °C) [The effect of the content of 3H5PhV in P with 4.1 mol % and 8.9 mol % 3H3PhP was similar to that of P(3HB) with ΔHm = 7.1 − 27.8 J·g−1, which is an index of the degree of crystallinity, suggesting that these samples still contained the P(3HB) crystal phase. However, these values are lower than those of other copolymers, such as P(3HB-3HA) with 6 mol % 3HA (C6–C8) with a Tm of 126 °C and ΔHm of 31 J·g−1 [Tm of 138 °C and ΔHm of 45 J·g−1 [Tgs of P(3HB-3H3PhP) and P(3HB-3H4PhB) were in the range of 9–20 °C and 7–10 °C, respectively, where these values are substantially higher than that of P(3HB) (5 °C) [Tg decreased as the 3H4MV content increased. The rigidity of the aromatic rings in the 3H3PhP and 3H4PhB monomers may account for these differences. The Tm of P(3HB-8 mol % 3H4PhB) is similar to that of low-density polyethylene, one of conventional plastics. The Tg of this PHAs (10–15 °C) is higher than that of low-density polyethylene (−30 °C), but it should be increased more to avoid secondary crystallization.The thermal properties of PHAs composed of 3HB and phenylalkanoic monomers were also determined ,17. The 31 J·g−1 , and P(3) (5 °C) , the Tg Tgs higher than 10 °C [Tm of 70 °C with small ΔHm (2.9 J·g−1), indicating that it is highly amorphous [ω-phenoxyalkanoic acid (C4–C8) did not show any crystalline melting endotherm, indicating that these polymers were totally amorphous [PHAs containing phenoxy groups are highly or totally amorphous with an 10 °C ,31,38,44morphous . In anotTg of 18 °C and Tm of 95 °C with a defined endothermic peak [para-position of the phenyl ring allows the polymer chains to form an ordered structure. In fact, P(3HA-15 mol % 3H5pMPhV) also showed a Tm of 60 °C and Tg of 14 °C. However, the ΔHm for this polymer is less than 1 J·g−1 [P(3H5pMPhV) containing the methylphenyl group as a side chain is a crystalline polymer with a mic peak , whereas 1 J·g−1 , indicat 1 J·g−1 , indicatpara-methylphenoxy group, is crystalline [Hm of P(3H4mMPhB-70 mol % 3H6mMPhHx) is 0.2 J·g−1, indicating low crystallinity. These results indicate that the position of methyl substitution has a profound effect on the crystallinity of the polymers. P(3H5PhV-6.7 mol % 3H4pMPxB-28.3 mol % 3H6pMPxHx), a PHA consisting of monomers containing methylphenoxy- and phenyl-groups, is amorphous [P(3H4pMPhB-71.5 mol % 3H6pMPhHx), a PHA consisting of monomers containing the stalline . Precipimorphous . This inortho,para-dinitrophenyl)valeric acid and NA, showed one Tm and two Tg peaks [Tgs indicates that the produced PHA is a mixture of two polymers [Tm of 56.42 °C and the Tg of −35.95 °C were assigned to the PHA produced from NA only, and the Tg of 28.74 °C is attributed to the newly synthesized polymer containing nitrophenyl rings. The lack of a defined Tm for this newly formed polymer indicates that the PHA containing the nitrophenyl group lacks crystalline domains and is thus amorphous.From DSC analysis, P(3HA-6.9 mol % 3H5opNPhV), a PHA produced from 5- led to a slight decrease in the Δm and Tm . This inTg at −21 °C that was not observed for the samples with 0–6.8 mol % of the 3H6pCPxHx unit. This indicates that the product with 19.6 mol % 3H6pCPxHx is not a random copolymer, but is heterogeneous, having chains and/or chain segments enriched with the 3H6pCPxHx repeating units. Additionally, these segments would have unique crystal structures as the original melting peak of the sample occurred at >64 °C, where no melting peak was observed for P(3HA).P(3HA-19.6 mol% 3H6pCPxHx) showed a new Tms of 52 °C (monofluorinated P(3H5pFPxV-8.7 mol % 3H7pFPxHp)) and 102 °C (difluorinated P(3H5opFPxV)) [Generally, fluoropolymers exhibit good thermal resistance. PHAs with a fluorinated side group also exhibit the expected behavior, with opFPxV)) . The widopFPxV)) . IncorpoTg of 4 °C with no clear melting peaks [P(3H5TPxV-3H7TPxHp), a PHA consisting of thiophenoxy monomer units, has a ng peaks , indicatTg was 36 °C and the Tm was 150 °C. These values are higher than those of other aromatic PHAs. Generally, a high molecular weight can lead to high Tg and/or Tm values, but mcl-PHA  and PHB , which have relatively high molecular weights, do not exhibit such high Tg or Tm values. Therefore, it was suggested that the incorporation of 79.8% of the 3H5BzV unit resulted in these high values. However, the thermal properties of other PHAs bearing benzoyl groups were similar to those of the other aromatic PHAs [The PHA containing 79.8% 3H5BzV and 3HA (as determined from the GC-MS peak area) is relatively hard at room temperature, which indicates that this polymer has characteristic thermal properties . From DStic PHAs . Thus, iTm and ΔHm decreased, whereas the Tg increased. This trend in the Tg is much different from the cases of aliphatic monomers. The relationship between the aromatic monomer content and the Tg has been studied detail [As described in d detail ,17.Tg of a copolymer (Tgr) can be calculated by applying the Fox equation, using the Tg of the constituent polymers (Tg1 and Tg2) [Tg values of the homopolymers are given in Kelvin, and W1 and W2 are the weight fractions of the respective polymers in the copolymer.The and Tg2) :1/Tgr = Tg = 280.9 K, W3H3PhP = 0), P(3HB-4.1 mol% 3H3PhP) , and P(3HB-8.9 mol% 3H3PhP) ), the Tg of the P(3H3PhP) homopolymer was estimated to be 62 °C (335 K). This estimated value is much higher than ambient temperature and is very close to that of poly(lactide) (Tg = 60 °C) [Tg for P(3HB-3H3PhP) with 33 mol% 3H3PhP (W3H3PhP = 0.45) is 30 °C, which is the same as the culture temperature for PHA production. PHAs with Tgs exceeding ambient temperature will be produced as rigid amorphous polymers. If PHA synthases do not prefer to polymerize rigid amorphous PHA, the production of P(3HB-3H3PhP) with 3H3PhP fractions of over 33 mol% may be a challenging task [In the literature , from thing task .Tg are shown in Tgs and those predicted from the Fox equation. With increasing alkyl chain length of the aromatic side group on the monomers bearing the phenyl group, the Tg of the PHAs tends to decrease. This may be the result of an increase in the free volume. The rigidity of the side group further hampers the motion of the polymer backbone. Similar trends have been reported for mcl-PHAs [Tg values from another study using the Fox equation were 60 °C for P(3H3PhP) and 27 °C for P(3H4PhB) [Tg than 3H4PhB. These results suggest that the Tg of aromatic PHAs bearing phenyl groups depends not only on the size of the side group, but also on its rigidness.The correlation between the average number of methylene units in the aromatic side chain and the mcl-PHAs , poly , which aTg values of aromatic PHAs bearing phenoxy, methylphenyl, methylphenoxy, and nitrophenyl groups followed trends similar to those of PHAs bearing the phenyl group.As shown in Tg increases as the aromatic monomer content increases, which differs from the behavior of aliphatic monomers. Even with the introduction of a small amount of aromatic monomers, crystallization of P(3HB) is strongly inhibited. This behavior is attributed to the rigidity of the aromatic rings in the side groups. Additionally, the degradation rate of aromatic PHAs is slower than that of mcl-PHAs. This suggests that aromatic PHAs could be used as a drug vehicle which achieve slow release of the encapsulated drug.The types of biosynthesized aromatic PHAs and their chemical and physical properties are summarized in this review. Various monomers containing phenyl, phenoxy, methylphenyl, methylphenoxy, nitrophenyl, nitrophenoxy, cyanophenoxy, fluorophenoxy, thiophenoxy, and benzoyl groups have been introduced into biosynthesized PHA chains. The chemical and physical properties of aromatic PHAs are different from those of aliphatic PHAs, and vary depending on the types of aromatic monomers. The Tg using the Fox equation is a useful tool for controlling the properties of PHAs. The composition of PHAs can be controlled by changing the ratio of the carbon sources and by modification of the metabolic pathway, including the β-oxidation cycle of the microorganisms involved in PHA production [The introduction of aromatic monomers broadens the range of applications by altering the properties of PHAs. Predicting the composition of PHAs that would provide the desired oduction ,39.Pseudomonas) that can polymerize aromatic monomers is a worthwhile undertaking. Reduction of the cost of the carbon sources and improving the productivity are critical approaches to reducing the production cost. The production of aromatic PHAs from biomass such as sugars without any precursor supplementation, which is generally expensive, would contribute to this end. However, the production of aromatic PHAs from sugars was just reported [For practical use of aromatic PHAs, certain issues must be addressed, such as the heterogeneity of the produced polymers, increasing the molecular weight of the PHAs, and reduction of the production cost. Under some conditions, aromatic PHAs are produced as heterogeneous polymers. Because the polymer homogeneity affects the material properties, the cultivation conditions should be optimized to produce homogeneous polymers rather than blend polymers. Additionally, increasing the molecular weight of PHAs is effective for improving the properties . If the reported ,18, so ireported .All aromatic PHAs described above have aromatic rings in their side chains. The production of PHAs having aromatic rings in their backbone has not been reported to date, and achieving this structure would be a challenging task because there is no PHA synthase capable of polymerizing aromatic rings in the PHA backbone thus far. If PHAs having aromatic rings in the backbone can be produced, they may show good thermostability, similar to polyethylene terephthalate.The incorporation of aromatic monomers is a promising method for improving the properties of PHAs and conferring physical properties superior to those of aliphatic PHAs. Further, this approach is not expected to compromise the important characteristics of PHAs, including their biocompatibility, biodegradability, and thermoplasticity. Thus, incorporation of aromatic monomers into the PHA chain is proposed as a promising method of improving the material properties of PHAs.


Mw > 106 g·mol−1) is an excellent precursor for high performance carbon fiber. In this work, a strategy for initiators for a continuous activator regeneration atom transfer radical polymerization (ICAR ATRP) system for acrylonitrile (AN) was firstly established by using CuCl2·2H2O as the catalyst and 2,2′-azobis(2-methylpropionitrile) (AIBN) as the thermal initiator in the presence of ppm level catalyst under ambient and high pressure (5 kbar). The effect of catalyst concentration and polymerization temperature on the polymerization behaviors was investigated. It is important that PAN with ultrahigh viscosity and average molecular weight  could be synthesized within 2 h under high pressure.It is well known that well-defined polyacrylonitrile (PAN) with high molecular weight ( In the past decades, we have witnessed great progress of ”living”/controlled radical polymerization (LRP) in terms of the ability of macromolecular design and precision synthesis. Especially atom transfer radical polymerization (ATRP) ,2,3,4, aMw > 1000 kg·mol−1) and narrow molecular weight distribution (Mw/M¬n = 1.03) polymethyl methylacrylate (PMMA) under 5 kbar. Fukuda and his partner 0:[CuCl2·2H2O]0:[TPMA]0:[AIBN]0 = 500:1:0.05:0.1:0.2 was carried out in a clean ampoule (10 mL) (under atmospheric pressure) or a bag made by polyperfluorinated ethylene propylene film (under high pressure). The reaction mixture was prepared by adding AN , BMPB2 , CuCl2·2H2O , TPMA , AIBN  and DMSO (3.0 mL). The solution was bubbled with argon for about 20 min to eliminate the dissolved oxygen. Then, the ampoule was flame-sealed and placed in an oil bath at 60 °C. The bag was sealed by plastic-envelop machine and placed in a water bath with high pressure (5 kbar). The high pressure reaction system (HPP. L2-600/0.6) was purchased from Tianjin Huatai Sen Miao Biological Engineering Technology Co. Ltd. . The reactor includes a hydraulic piston-cylinder unit with pressure reaction vessel equipped with a temperature controller and a pressure sensor using water as the medium of pressure conductivity. The ampoule or a bag was opened after the desired polymerization time under atmospheric pressure and high pressure, respectively. The mixture was dissolved in a certain amount of DMF and precipitated into 250 mL of methanol. The polymers were isolated by suction filtering in a Buchner funnel and dried in vacuum oven until a constant weight at 35 °C. The monomer conversion was obtained gravimetrically.A typical ICAR ATRP procedure with the molar ratio of [AN]1H NMR spectra of the obtained polymers were recorded on a Bruker 300 MHz nuclear magnetic resonance instrument using CDCl3 as the solvent and tetramethylsilane (TMS) as an internal standard. The number-average molecular weight  and molecular weight distribution (Mw/Mn) values of the resultant polymers determined using a TOSOH HLC-8320 gel permeation chromatograph (GPC) equipped with a refractive-index detector , using TSKgel guard column SuperMP-N (4.6 mm × 20 mm) and two TSK gel SupermultiporeHZ-N (4.6 mm × 150 mm) with measurable molecular weight ranging from 102 to 106 g/mol. DMF with 0.5 mmol/L of LiBr was used as eluent at a flow rate of 1.0 mL/min at 40 °C. GPC samples were injected using a TOSOH plus autosampler and calibrated with PMMA standards purchased from TOSOH.The Mw/Mn = 1.20, 250 ppm catalyst, Entry 1 in Mw/Mn = 1.80 -catalyzed ICAR ATRP was firstly investigated under ambient pressure, and the results are shown in aszewski ) were adIt is well known that temperature plays a vital role in controlled polymerization systems. As shown in Mn,GPC/2.5) close to the corresponding theoretical values and narrower molecular weight distribution (Mw/Mn < 1.20) with the monomer conversion  prepared by ICAR ATRP under ambient pressure was analyzed by 1H NMR spectroscopy as shown in 2, respectively. Moreover, the peaks of d (δ = 3.20 ppm) and c (δ = 2.11 ppm) corresponded to the methylene protons and methyl protons in the PAN backbone. Because of the electron-attracting function of the Br atom, the signals of peak d′ (δ = 5.21~5.32 ppm) and c′ (δ = 4.15 ppm) were attributed to the methylene protons and methyl protons of the last repeat unit in the PAN chains. All of the above results confirmed the “living” feature of this ICAR ATRP system.In order to verify the “living” feature of this polymerization system, polymerization kinetics of Cu(II)-catalyzed ICAR ATRP of AN under ambient pressure were investigated as depicted in nversion b were ob2  was used to conduct Cu(II)-catalyzed ICAR ATRP under ambient and high pressure, respectively, and the results are shown in −1 (Mw/Mn = 1.20). By comparison, under high pressure (5 kbar), the monomer conversion could reach up to 72.3% within 2 h (Entry 4 in Mη) were determined by the Mark–Houwink equation [-1 was obtained with the large molar ratio of [AN]0:[BMPB2]0 = 20,000:1. Obviously, the high pressure polymerization technique would be more suitable for ultrahigh molecular weight polymer synthesis as compared to polymerization under atmosphere.In order to obtain high molecular weight PAN, a large molar ratio of AN to BMPBequation . A high 2·2H2O as the catalyst, TPMA as the efficient ligand, and BMPB2 as the difunctional initiator was successfully established in the presence of ppm level catalyst. In addition, PAN with an ultrahigh viscosity-average molecular weight up to Mη = 1,034,500 g·mol−1 could be synthesized just within 2 h under high pressure by Cu(II)-catalyzed ICAR ATRP of AN. It is particularly noteworthy that an enormous advantage in terms of the polymerization rate under high pressure in comparison with that under atmosphere was displayed. Therefore, the high pressure polymerization technique applied to ultrahigh molecular weight polymer synthesis is promising.A “living”/controlled radical polymerization system for ICAR ARTP of AN using CuCl