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Surface ligand engineering is of great importance for the preparation of one-dimensional (1D) CsPbBr3 nanowires for high-performance photodetectors. The traditional long-chain terminated ligands such as oleylamine/oleic acid (C18) used in the preparation of CsPbBr3 nanowires will form an electrically insulating layer on the surface of the nanowires, which hinders the effective transport of charge carriers in optoelectronic devices. In this paper, short-chain ligands, including dodecylamine/dodecanoic acid (C12), octylamine/octanoic acid (C8) and hexylamine/hexanoic acid (C6), are introduced to partially replace long-chain ligands (C18) to successfully prepare various CsPbBr3 nanowires via a solvothermal method. Microstructure characterization indicates that the four kinds of nanowires before/after surface ligand engineering, which are named as C18-CsPbBr3, C12/18-CsPbBr3, C8/18-CsPbBr3 and C6/18-CsPbBr3, all have high aspect ratio and purity. As compared with CsPbBr3 with long-chain terminated ligands, the C8/18-CsPbBr3 and C6/18-CsPbBr3 nanowires with shorter chain ligands exhibit superior photoluminescence (PL) performance and stability under adverse conditions such as ultraviolet irradiation and high temperature. The constructed photodetectors based on C8/18-CsPbBr3 and C6/18-CsPbBr3 nanowires have shown improved performances. This work provides a new idea for the preparation of CsPbBr3 nanowires with high optical properties, stability and charge transport, and the prepared CsPbBr3 nanowires have potential application prospects in optoelectronic devices.

The development of ultra-sensitive methods for detecting anions is limited by their low charge to radius ratios, microenvironment sensitivity, and pH sensitivity. In this paper, a magnetic sensor is devised that exploits the controllable and selective coordination that occurs between a magnetic graphene quantum dot (GQD) and fluoride anion (F–). The sensor is used to measure the change in relaxation time of aqueous solutions of magnetic GQDs in the presence of F‒ using ultra-low-field (118 μT) nuclear magnetic resonance relaxometry. The method was optimized to produce a limit of detection of 10 nmol/L and then applied to quantitatively detect F– in domestic water samples. More importantly, the key factors responsible for the change in relaxation time of the magnetic GQDs in the presence of F‒ are revealed to be the selective coordination that occurs between the GQDs and F‒ as well as the localized polarization of the water protons. This striking finding is not only significant for the development of other magnetic probes for sensing anions but also has important ramifications for the design of contrast agents with enhanced relaxivity for use in magnetic resonance imaging.

Gossypol, a toxic polyphenol extracted from cotton seeds, is hazardous to human and animal health. Taurine is considered as an essential or semi-essential amino acid and has diverse cytoprotective effects. This study was aimed to investigate the protective effect and molecular mechanism of taurine against apoptosis of C2C12 mouse myoblasts induced by gossypol. C2C12 mouse myoblasts were exposed to gossypol (0, 1 nM, 10 nM, 100 nM, 1 μM, and 10 μM). Cell numbers were rapidly decreased with increasing concentrations of gossypol. Gossypol significantly induced apoptosis, decreased Bcl2 expression, and increased the protein levels of Bax and the cleaved caspase 3. Taurine (0.24 mM) treatment largely rescued the cell number decreased by gossypol, attenuated gossypol-induced cell apoptosis. GPR87 knockdown abolished the inhibition by taurine of cell apoptosis. Furthermore, GPR87 overexpression attenuated cell apoptosis induced by gossypol. Both taurine treatment and GPR87 overexpression stimulated AKT phosphorylation but inhibited AMPK phosphorylation, whereas gossypol had the opposite effects. Taurine treatment promoted GPR87 expression and subcellular localization and partially rescued the inhibition of gossypol on this expression. In summary, these data reveal that taurine attenuates gossypol-induced apoptosis of C2C12 mouse myoblasts via the GPR87-AMPK/AKT signaling.

Avian leukosis virus subgroup J (ALV-J) is an important pathogen for various neoplasms and causes significant economic losses in the poultry industry. Serological detection of specific antibodies against ALV-J infection is important for successful clinical diagnosis. Here, a 293F stable cell line was established to stably express gp85 protein. In this cell line, gp85 protein was expressed at approximately 30 mg/L. A subgroup-specific indirect enzyme-linked immunosorbent assay (iELISA) was developed using ALV-J gp85 protein as coated antigen to detect antibodies against ALV-J. The sensitivity of the iELISA (1:51200 diluted in serum) was 16 times more than that of indirect immunofluorescence assay (IFA; 1:3200 diluted in serum). Moreover, there was no crossreactivity with antibodies against other common avian viruses and other avian leukosis virus subgroups, such as subgroups A and B. The practicality of the iELISA was further evaluated by experimental infection and clinical samples. The results from experimental infection indicated that anti-ALV-J antibodies were readily detected by iELISA as early as 4 weeks after ALV-J infection, and positive antibodies were detected until 20 weeks, with an antibody-positive rate of 11.1% to 33.3%. Moreover, analysis of clinical samples showed that 9.49% of samples were positive for anti-ALV-J antibodies, and the concordance rate of iELISA and IFA was 99.24%. Overall, these results suggested that the subgroup-specific iELISA developed in this study had good sensitivity, specificity, and feasibility. This iELISA will be very useful for epidemiological surveillance, diagnosis, and eradication of ALV-J in poultry farms.

All-inorganic CsPbX3 (X = Cl, Br, I) perovskite quantum dots (QDs) have great potential for various applications due to their excellent photoluminescence properties. However, poor stability under long-term storage hinders their applications. Herein we report the utilization of porous boron nitride nanofibers (BNNFs) as a promising carrier for anchoring of CsPbBr3 QDs. Due to the good dispersion and immobilization of CsPbBr3 QDs, the resulting CsPbBr3/BNNF composites show excellent photostability and superior long-term storage stability in an air environment. Moreover, the CsPbBr3/BNNF composites exhibit an interesting ammonia-responsive behavior: i.e., a distinct decrease in photoluminescence intensity upon exposure to ammonia gas and the subsequent photoluminescence recovery after post-treatment in nitrogen gas. Even after treatment with ammonia gas for 3 h, the composites can still be recovered under nitrogen gas treatment. The fast response, reversibility, and stability of CsPbBr3/BNNF composites in the presence of ammonia gas could inspire a broader range of applications.

Jasmonic acid (JA)- and ethylene-mediated signaling pathways are reported to have synergistic effects on inhibiting gray mold. The present study aimed to explain the role of ethylene perception in methyl jasmonate (MeJA)-mediated immune responses. Results showed that exogenous MeJA enhanced disease resistance, accompanied by the induction of endogenous JA biosynthesis and ethylene production, which led to the activation of the phenolic metabolism pathway. Blocking ethylene perception using 1-methylcyclopropene (1-MCP) either before or after MeJA treatment could differently weaken the disease responses induced by MeJA, including suppressing the induction of ethylene production and JA contents and reducing activities of lipoxygenase and allene oxide synthase compared to MeJA treatment alone. Consequently, MeJA-induced elevations in the total phenolic content and the activities of phenylalanine ammonia-lyase, cinnamate 4-hydroxylase, 4-coumarate:coenzyme A ligase, and peroxidase were impaired by 1-MCP. These results suggested that ethylene perception participated in MeJA-mediated immune responses in tomato fruit.

In South-East Asia, cocoa production is dramatically affected by cocoa pod borer (CPB) infestations. As an alternative tool to chemical control, the efficacy of attract-and-kill strategy (CPB sex-p...

Subgroup J avian leukemia virus (ALV-J), belonging to the genus Alpharetrovirus, enters cells through its envelope surface unit (gp85) via specifically recognizing the cellular receptor chicken Na(+)/H(+) exchanger type I (chNHE1), the 28 to 39 N-terminal residues of which were characterized as the minimal receptor functional domain in our previous studies. In this study, to further clarify the precise organization and properties of the interaction between ALV-J gp85 and chNHE1, we identified the chNHE1-binding domain of ALV-J gp85 using a series of gp85 mutants with segment substitutions and evaluating their effects on chNHE1 binding in protein-cell binding assays. Our results showed that hemagglutinin (HA) substitutions of amino acids (aa) 38 to 131 (N terminus of gp85) and aa 159 to 283 (C terminus of gp85) significantly inhibited the interaction between gp85 and chNHE1/chNHE1 loop 1. In addition, these HA-substituted chimeric gp85 proteins could not effectively block the entry of ALV-J into chNHE1-expressing cells. Furthermore, analysis of various N-linked glycosylation sites and cysteine mutants in gp85 revealed that glycosylation sites (N6 and N11) and cysteines (C3 and C9) were directly involved in receptor-gp85 binding and important for the entry of ALV-J into cells. Taken together, our findings indicated that the bipartite sequence motif, spanning aa 38 to 131 and aa 159 to 283, of ALV-J gp85 was essential for binding to chNHE1, with its two N-linked glycosylation sites and two cysteines being important for its receptor-binding function and subsequent viral infection steps. IMPORTANCE Infection of a cell by retroviruses requires the attachment and fusion of the host and viral membranes. The specific adsorption of envelope (Env) surface proteins to cell receptors is a key step in triggering infections and has been the target of antiviral drug screening. ALV-J is an economically important avian pathogen that belongs to the genus Alpharetrovirus and has a wider host range than other ALV subgroups. Our results showed that the amino acids 38 to 131 of the N terminus and 159 to 283 of the C terminus of ALV-J gp85 controlled the efficiency of gp85 binding to chNHE1 and were critical for viral infection. In addition, the glycosylation sites (N6 and N11) and cysteines (C3 and C9) of gp85 played a crucial role in the receptor binding and viral entry. These findings might help elucidate the mechanism of the entry of ALV-J into host cells and provide antiviral targets for the control of ALV-J.

Isoflavones possess a wide range of physiological effects. However, it is still unclear whether isoflavones can promote milk synthesis in mammary gland. This study aimed to determine the effects of a main soy isoflavone, daidzein, on milk synthesis and proliferation of mammary epithelial cells (MECs) and reveal the underlying molecular mechanism. Primary bovine MECs were treated with different concentrations of daidzein (0, 5, 10, 20, 40, and 80 μM). Daidzein dose-dependently promoted α- and β-casein and lipid synthesis, cell cycle transition, and cell amount, with the best stimulatory effect at 20 μM. Daidzein also stimulated mTOR activation and Cyclin D1 and SREBP-1c expression. Daidzein induced the expression and nuclear localization of estrogen receptor α (ERα), and ERα knockdown blocked the stimulation of daidzein on these above signaling pathways. ERα knockdown also abolished the stimulation of daidzein on NFκB1 expression and phosphorylation, and NFκB1 was required for daidzein to enhance the mTOR, Cyclin D1 and SREBP-1c signaling pathways. In summary, our findings reveal that daidzein stimulates milk synthesis and proliferation of MECs via the ERα-dependent NFκB1 activation.

Taurine, a β-aminosulfonic acid, exerts many cellular physiological functions. It is still unknown whether taurine can regulate milk synthesis in the mammary gland. Therefore, in this study we investigated the effects and mechanism of taurine on milk synthesis in mammary epithelial cells (MECs). Bovine MECs (BMECs) cultured in FBS-free OPTI-MEMImedium were treated with taurine (0, 0.08, 0.16, 0.24, 0.32, and 0.4 mM). Taurine treatment led to increased milk protein and fat synthesis, mTOR phosphorylation, and SREBP-1c protein expression, in a dose-dependent manner, with an apparent maximum at 0.24 mM. Gene function study approaches revealed that the GPR87-PI3K-SETD1A signaling was required for taurine to increase the mTOR and SREBP-1c mRNA levels. Taurine stimulated GPR87 expression and cell membrane localization in a dose dependent manner, suggesting a sensing mechanism of GPR87 to extracellular taurine. Collectively, these data demonstrate that taurine promotes milk synthesis via the GPR87-PI3K-SETD1A signaling.

Previous studies have implicated that purine-rich element binding protein B (PURB) is a key regulator of gene transcription and cell physiology. Whether PURB plays a regulatory role in milk synthesis in bovine mammary epithelial cells (BMECs) is not known. We observed that Met and Leu increased PURB expression and nuclear localization. Overexpression of PURB led to increased milk protein and fat synthesis as well as mTOR and SREBP-1c expression whereas PURB knockdown had the opposite effects. The PI3K inhibitor wortmannin totally abolished the stimulation of Met and Leu on PURB expression, and we further confirmed that PURB was required for Met and Leu to stimulate mTOR phosphorylation and SREBP-1c expression. We also demonstrated that PURB binds to the promoters of mTOR and SREBP-1c, and these bindings were increased by Met and Leu stimulation. In summary, our data reveal that PURB is required for amino acids to stimulate mTOR and SREBP-1c gene expression, and PURB is a positive regulator of amino acid-induced PI3K-regulated milk protein and fat synthesis in BMECs.

The intracellular fatty acid-binding proteins (FABPs) are a well-conserved family that function as lipid chaperones. Ongoing studies are focused on identification of the mechanistic complexity and vast biological diversity of different isoforms of FABPs. However, the molecular mechanism of FABP5 in the regulation of milk fat synthesis in the mammary gland of dairy cows is still largely unknown. Here, we report that FABP5 acts as a critical regulator of terol response element-binding protein-1c (SREBP-1c) gene expression induced by methionine (Met) and estrogen (E2) in bovine mammary epithelial cells (BMECs). We observed that the expression of FABP5 was markedly higher in dairy cow mammary tissue during the lactating period than the puberty period and the dry period. FABP5 is located in the cytoplasm, and Met and E2 significantly increase the protein levels of FABP5 in BMECs. Using gene function study approaches, we revealed that FABP5 positively regulates SREBP-1c gene expression and promotes milk fat synthesis. We confirmed that FABP5 is required for Met- and E2-induced SREBP-1c gene expression and milk fat synthesis. We further uncovered that fatty acids are needed for FABP5-mediated SREBP-1c gene expression. Thus, our study demonstrates that FABP5 is a critical regulator of Met- and E2-induced SREBP-1c gene expression leading to milk fat synthesis.

It remains unclear how the biomass and biological nitrogen (N) fixation by lucerne (Medicago sativa) respond to different irrigations and sowing modes in arid and semi-arid regions. To clarify the question, a field 15N isotope labeling experiment was conducted to evaluate the biological N fixation in pure and mixed lucerne (lucerne growing alone or lucerne growing with Elymus nutans (EN), Agropyron cristatum (AC) or Bromus inermis (BI)) under four water conditions: a control (CK) and three levels of irrigation (low (LI), medium (MI), and high (HI)) by the 15N dilution technique. The biomass of lucerne in pure and mixed lucerne conditions responded nonlinearly to different irrigations, and in mixed lucerne, the biomass varied with the specific coexisting plant species, with the maximum in CK for lucerne-EN, in LI for lucerne-AC, and in HI for lucerne-BI. The N derived from the atmosphere (%Ndfa) in mixed lucerne (90.8%) was higher than that in pure lucerne (88.3%). Biological N fixation by lucerne in mixed lucerne ranged from 2.86 to 15.05 g N m−2, and that in pure lucerne was approximately 10.99 g N m−2. A positive relation was observed between the competitive response and N fixation by lucerne, which showed that interspecific competition or facilitation in mixed lucerne altered N fixation by lucerne. These findings indicate that an appropriate legume-grass mixture with moderate irrigation can promote the biomass of forages and biological N fixation by legumes and further improve soil quality in artificial grasslands.

To assess the diagnostic performance of the calcification remodeling index (RI) as assessed by coronary computed tomography angiography (coronary CTA) to predict the presence of severe coronary stenosis in atherosclerotic coronary lesions with moderate to severe calcification.Patients who underwent coronary CTA and invasive coronary angiography (ICA) within one month and had moderately to severely calcified lesions as revealed by coronary CTA, were retrospectively included. The calcification RI was calculated as the ratio of the cross-sectional lumen area (with inclusion of calcium area) of the most severely calcified site to the proximal reference lumen area. Other parameters, such as the calcium volume, regional Agatston score, calcification length, involved calcium arc quadrants and CTA-assessed diameter stenosis, were also recorded. A multivariate model was used to identify the variables that predict the presence of severe coronary stenosis (diameter stenosis ≧ 70%) as determined by ICA.422 patients with 629 lesions were finally included in the study. Lesions with severe stenoses as determined by ICA tended to have larger calcium volumes, regional Agatston scores, CTA-assessed diameter stenoses, longer calcium length, more involved calcium arc quadrants and a significantly smaller calcification remodeling index. ROC curve analysis determined the best cutoff value of the calcification RI as 0.94 (AUC = 0.816, p 0.001), which yielded highest diagnostic accuracy (83.3%, 524/629) to identify severe coronary stenosis. Among all parameters, calcification RI ≦0.94 is the strongest independent predictor (odds ratio: 17.5, p 0.001) of severe coronary stenosis.With an optimalcut-off value of 0.94, calcification RI is the strongest independent predictor of severe coronary stenosis in calcified coronary atherosclerotic lesions.

The design of contrast agents (CAs) with high magnetic relaxivities is a key issue in the field of magnetic resonance imaging (MRI). The traditional strategy employed is aimed at optimizing the structural design of the magnetic atoms in the CA. However, it is difficult to obtain an agent with magnetic relaxivity over 100 mM−1 s−1 using this approach. In this work, we demonstrate that modulation of the localized superacid microenvironment of certain CAs (Gd3+ loaded polyethylene glycol modified graphene oxide quantum dots or ‘GPG’ for short) can effectively enhance the longitudinal magnetic relaxivities (r1) by accelerating proton exchange. r1 values of a series of GPGs are significantly increased by 20–30 folds compared to commercially available CAs over a wide range of static magnetic field strengths (e.g. 210.9 mM−1 s−1 vs. 12.3 mM−1 s−1 at 114 μT, 127.0 mM−1 s−1 vs. 4.9 mM−1 s−1 at 7.0 T). GPG aided MRI images is then acquired both in vitro and in vivo with low biotoxicities. Furthermore, folic-acid-modified GPG is demonstrated suitable for MRI-fluorescence dual-modal tumor targeting imaging in animals with more than 98.3% specific cellular uptake rate.

Glycyl-tRNA synthetase (GlyRS) has non-canonical roles beyond aminoacylation, but the molecular mechanism is largely unknown. We have previously found that GlyRS is phosphorylated in the cytoplasm of bovine mammary epithelial cells (bMECs) in response to amino acid stimulation, and the phosphorylated GlyRS enters nucleus to stimulate gene expression for milk synthesis. In this study, we aim to uncover the upstream kinase of GlyRS and reveal the signaling pathways that methionine (Met) stimulates GlyRS phosphorylation. We show that mitogen-activated protein kinase 10 (MAP3K10) interacts with GlyRS in bMECs by Co-IP, mass spectrometry, and Western blotting analysis. We further identify that MAP3K10 is an upstream kinase of GlyRS by in vitro kinase assay and MAP3K10 stimulates NFκB1 phosphorylation via activating GlyRS. We also uncover that Met stimulates GlyRS phosphorylation via the GPR87-CDC42/Rac1-MAP3K10 signaling pathway. Our findings help to understand the molecular mechanism of GlyRS in cellular signaling transduction.

Semen Vaccariae, the seed of Vaccaria segetalis, is traditionally used in East Asian countries for the treatment of breast milk deficiency, but the underlying molecular mechanism has not been discovered yet. The present study assessed the stimulatory effect of vaccarin, one of the major constituents of Semen Vaccariae, on proliferation of and milk synthesis in bovine mammary epithelial cells (BMECs) and explored the corresponding molecular mechanism. Vaccarin affected cell proliferation and milk fat and protein synthesis in a concentration-dependent manner, with the best stimulatory effects at 0.5 μg/ml concentration. Vaccarin (0.5 μg/ml) had the similar effects as prolactin (Prl, 0.5 μg/ml) on cell proliferation, milk fat and protein synthesis, expression of Cyclin D1, phosphorylation of mechanistic target of rapamycin (mTOR), and expression and maturation of sterol regulatory element binding protein 1c (SREBP-1c). Vaccarin stimulated these signaling pathways via the Prl receptor-phosphatidyl inositol 3-kinase (PI3K) signaling. Vaccarin also concentration-dependently stimulated expression of the Prl receptor, with the best effects at 0.5 μg/ml concentration. In summary, we demonstrate that vaccarin promotes proliferation of and milk synthesis in BMECs through the Prl receptor-PI3K signaling, suggesting that vaccarin might be the main active component promoting milk production of BMECs in Semen Vaccariae.

Amino acids can stimulate milk fat synthesis, but the underlying molecular mechanism is still largely unknown. In this study, we studied the regulatory role and corresponding molecular mechanism of cAMP response element-binding protein-regulated transcription coactivator 2 (CRTC2) in amino acid-induced milk fat synthesis in mammary epithelial cells. We showed that leucine and methionine stimulated CRTC2 but not p-CRTC2(Ser171) expression and nuclear localization in cow mammary epithelial cells. Knockdown of CRTC2 decreased milk fat synthesis and sterol regulatory element binding protein 1c (SREBP-1c) expression and activation, whereas its overexpression had the opposite effects. Neither knockdown nor overexpression of CRTC2 affected β-casein synthesis and phosphorylation of the machanistic target of rapamycin (mTOR), suggesting that CRTC2 only regulates milk fat synthesis. CRTC2 knockdown abolished the stimulation of leucine and methionine on SREBP-1c expression and activation. Knockdown or overexpression of CRTC2 did not affect the protein level of cAMP-response element-binding protein (CREB) and its phosphorylation but decreased or increased the binding of p-CREB to the promoter of SREBP-1c gene and its mRNA expression, respectively. Mutation of Ser171 of CRTC2 did not alter the stimulation of CRTC2 on SREBP-1c expression and activation, further suggesting that CRTC2 functions in the nucleus. mTOR inhibition by rapamycin totally blocked the stimulation of leucine and methionine on CRTC2 expression. The expression of CRTC2 was dramatically higher in the mouse mammary gland of lactation period, compared with that of the dry and puberty periods, whereas p-CRTC2(Ser171) was not changed, further supporting that CRTC2 is a key transcription coactivator for milk fat synthesis. These results uncover that CRTC2 is a key transcription coactivator of amino acid-stimulated mTOR-mediated milk fat synthesis in mammary epithelial cells.

The free amino acids play an important role in total nitrogen (N) absorption for plants. Most previous studies, however, only focused on the organic N uptake of glycine or several simple amino acids by plants. The purpose of this research was to quantify the uptake rate of N derived from multiple amino acids and evaluate the importance of different N forms (amino acid N and inorganic N) to plant nutrition. A range of amino acids (aspartic acid, lysine, leucine, phenylalanine, proline, valine, alanine, glutamine, isoleucine and glycine) and inorganic N (NH4+ and NO3-) with the same amount of N for each of 12 N forms were used in a short-term (3 h) 15N labeling experiment in a temperate grassland of North China. Two dominant plant species, Stipa grandis and L. chinensis, were investigated. Within 3 h, the N derived from multiple amino acids were both absorbed by S. grandis and L. chinensis, contributed about 25% and 37% to the total N uptake by two plant species, respectively. Leymus chinensis is relatively more effective in utilizing N derived from amino acids compared to S. grandis, while S. grandis takes up more inorganic N of NH4+ and NO3-. The uptake rates of N derived from multiple amino acids by S. grandis and L. chinensis differed greatly, and which have significant positive relations with soil amino acid concentrations (r2 = 0.59, P

We report the synthesis of Fe3O4 nanoparticles (NPs)-decorated porous boron nitride nanofibers (BNNFs) as a novel BNNF@Fe3O4 nanocomposite via a solvothermal method. One-dimensional porous BNNFs with high porosity are utilized as carrier for supporting and immobilization of Fe3O4 NPs with average diameters of ∼7 nm. Magnetic measurement confirms that nanocomposites are in superparamagnetic state. The nanocomposites with even sparsely loaded Fe3O4 NPs can be easily operated in a low magnetic field. Furthermore, the BNNF@Fe3O4 nanocomposites with sparsely loaded Fe3O4 NPs exhibit remarkable adsorption effect on Pb(II) ions in accordance with the Langmuir and the pseudo-second-order kinetic models. The synergistic adsorption of Pb(II) on BNNF@Fe3O4 nanocomposites are further studied via the adsorption of Pb(II) using BN nanocomposites with different loading amount of Fe3O4 NPs. The results indicate that the strong electrostatic interaction between BNNFs and Pb(II) results in the initial fast adsorption of Pb(II) on the surface of BNNFs, and the weak electrostatic force of Fe3O4 leads to the subsequent adsorption of Pb(II) on Fe3O4, showing an interesting component-ratio related synergistic effect. The synergistic adsorption mechanism clarified in this study will advance the fundamental knowledge and scientific understanding of the nanocomposite adsorbents.

Amino acids are required for the activation of mammalian target of rapamycin (mTOR) to increase cell growth, protein and lipid synthesis, and inhibit autophagy. However, the mechanism through which amino acids activate the mTOR signaling is still largely unknown. In our previous study, we discovered that glycyl-tRNA synthetase (GlyRS) is a key mediator of amino-acid-induced mTOR expression and activation in bovine mammary epithelial cells (BMECs). Here we show that amino acids stimulate GlyRS nuclear localization for mTOR expression in BMECs. Met stimulates GlyRS nuclear localization, and the nuclear GlyRS is cleaved into a C-terminus-containing truncated form. We prove that GlyRS has a bipartite nuclear leading sequences, and GlyRS is phosphorylated at Thr544 and Ser704 in the cytoplasm under the stimulation of amino acids (Met, Leu, and Lys). The nuclear GlyRS physically binds to nuclear factor kappa B1, triggers its phosphorylation, thereby enhancing mRNA expression of its target genes including mTOR, S6K1, and 4EBP1. We further demonstrate that GlyRS is required for the inhibition of autophagy by Met. Thus our work elucidates that amino acids trigger GlyRS phosphorylation and nuclear localization to enhance the mRNA expression of mTOR.

The ever-growing market of electrochemical energy storage impels the advances on cost-effective and environmentally friendly battery chemistries. Lithium-ion batteries (LIBs) are currently the most critical energy storage devices for a variety of applications, while sodium-ion batteries (SIBs) are expected to complement LIBs in large-scale applications. In respect to their constituent components, the cathode part is the most significant sector regarding weight fraction and cost. Therefore, the development of cathode materials based on Earth’s abundant elements (Fe and Mn) largely determines the prospects of the batteries. Herein, we offer a comprehensive review of the up-to-date advances on Fe- and Mn-based cathode materials for LIBs and SIBs, highlighting some promising candidates, such as Li- and Mn-rich layered oxides, LiNi0.5Mn1.5O4, LiFe1-xMnxPO4, NaxFeyMn1-yO2, Na4MnFe2(PO4)(P2O7), and Prussian blue analogs. Also, challenges and prospects are discussed to direct the possible development of cost-effective and high-performance cathode materials for future rechargeable batteries.

Visible-light-promoted hydrolysis of silanes catalyzed by (TAP)Rh–I to produce silanols and dihydrogen efficiently under mild conditions was reported. (TAP)Rh–H was observed as the key intermediate through stoichiometric activation of the Si–H bond by (TAP)Rh–I. Addition of water drove the stoichiometric activation of Si–H into catalysis.

A new technique of carbon capture by ammonia with the reinforced crystallization was put forward in this paper in view of existing problems. Through the application of low concentration of ammonia with alcohols added in to reinforce crystallization process, ammonia escapes less, and the load capacity increases and at the same time, low energy consumption of regeneration could be achieved easily. By experiments and theoretical calculation, it was found that the absorbent capacity could reach 1.64 kgCO 2 /kgNH 3 , and ammonia escape at the outlet could be neglected. What's more, through rough estimation, thermal energy consumption for regeneration was only about 1/3 as much as that of conventional rich solution regeneration. In conclusion, the new technique is an improvement with great advantages for carbon capture by ammonia.

Chicken Na + /H + exchanger type I (chNHE1), a multispan transmembrane protein, is a cellular receptor of the subgroup J avian leukosis virus (ALV-J). To identify the functional determinants of chNHE1 responsible for the ALV-J receptor activity, a series of chimeric receptors was created by exchanging the extracellular loops (ECL) of human NHE1 (huNHE1) and chNHE1 and by ECL replacement with a hemagglutinin (HA) tag. These chimeric receptors then were used in binding and entry assays to map the minimal ALV-J gp85-binding domain of chNHE1. We show that ECL1 of chNHE1 (chECL1) is the critical functional ECL that interacts directly with ALV-J gp85; ECL3 is also involved in ALV-J gp85 binding. Amino acid residues 28 to 39 of the N-terminal membrane-proximal region of chECL1 constitute the minimal domain required for chNHE1 binding of ALV-J gp85. These residues are sufficient to mediate viral entry into ALV-J nonpermissive cells. Point mutation analysis revealed that A30, V33, W38, and E39 of chECL1 are the key residues mediating the binding between chNHE1 and ALV-J gp85. Further, the replacement of residues 28 to 39 of huNHE1 with the corresponding chNHE1 residues converted the nonfunctional ALV-J receptor huNHE1 to a functional one. Importantly, soluble chECL1 and huECL1 harboring chNHE1 residues 28 to 39 both could effectively block ALV-J infection. Collectively, our findings indicate that residues 28 to 39 of chNHE1 constitute a domain that is critical for receptor function and mediate ALV-J entry. IMPORTANCE chNHE1 is a cellular receptor of ALV-J, a retrovirus that causes infections in chickens and serious economic losses in the poultry industry. Until now, the domains determining the chNHE1 receptor function remained unknown. We demonstrate that chECL1 is critical for receptor function, with residues 28 to 39 constituting the minimal functional domain responsible for chNHE1 binding of ALV-J gp85 and efficiently mediating ALV-J cell entry. These residues are located in the membrane-proximal region of the N terminus of chECL1, suggesting that the binding site of ALV-J gp85 on chNHE1 is probably located on the apex of the molecule; the receptor-binding mode might be different from that of retroviruses. We also found that soluble chECL1, as well as huECL1 harboring chNHE1 residues 28 to 39, effectively blocked ALV-J infection. These findings contribute to a better understanding of the ALV-J infection mechanism and also provide new insights into the control strategies for ALV-J infection.

The control of the polymerization of a wide range of monomers under mild conditions by a single catalyst remains a major challenge in polymer science. We report a versatile, well-defined organocobalt salen complex to control living radical polymerization of different categories of monomers, including acrylates, acrylamides and vinyl acetate, under visible light irradiation at ambient temperature. Both household light and sunlight were effectively applied in the synthesis of polymers with controlled molecular weights and narrow polydispersities. Narrowly dispersed block copolymers (Mw/Mn < 1.2) were obtained under various conditions. The structures of the polymers were analyzed by 1H NMR, 2D NMR, 13C NMR, GPC, MALDI-TOF-MS and isotopic labeling experiments, which showed that the ω and α ends of the polymer chains were capped with (salen)Co and -CO2CH3 segments, respectively, from the photoinitiator (salen)Co-CO2CH3. The ω end was easily functionalized through oxygen insertion followed by hydrolysis from 18O2 to -18OH. This robust system can proceed without any additives, and offers a versatile and green way to produce well-defined homo and block copolymers.

Visible light induced living radical polymerization of a wide scope of acrylates mediated by organocobalt porphyrins was developed. The photocleavage of the Co–C bond of organocobalt porphyrin produced carbon centered radicals, which initiated polymerization, and porphyrin cobalt(II), a persistent metal-centered radical. The organocobalt porphyrins were highly sensitive to external visible light irradiation so that photostimulus was used to control the initiation steps and regulate chain growth by reversibly activating the Co–C bond. Polymerization occurred spontaneously under irradiation and stopped promptly once shutting down light source. The scope of monomers was successfully extended from acrylamides to various hydrophobic and hydrophilic acrylates via the control of the light intensity. The structure of polyacrylate obtained was confirmed by 2D NMR, 13C NMR, GPC, and MALDI-TOF-MS. One of the unique features of this neat visible light induced polymerization process is that organocobalt porphyrins pla...

BACKGROUND: Mitogen-activated protein kinase (MAPK, EC 2.7.11.24) cascade from several plant species has been shown to be activated during response to abiotic stress. Ethylene plays an important role in fruit tolerance to environmental stress. However, the mechanisms by which MAPK regulates defence systems in fruit and the relationship between MAPK and ethylene remain to be determined. RESULTS: MAPK inhibitor significantly decreased the chilling tolerance of tomato (Lycopersicon esculentum cv. Lichun) fruit during cold storage. Moreover, decreases in ethylene content, LeACS2 expression and activities of 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (ACS, EC 4.4.1.14) and ACC oxidase (ACO, EC 1.14.17.4) due to MAPK inhibitor occurred within 24 h after cold treatment. Upon treatment with cold and ethephon, the ethylene content, activities of ACS and ACO and expression of LeACS2, LeACO1 and LeMAPK4 increased. CONCLUSION: The results showed the regulation of MAPK in ethylene biosynthesis to protect tomato fruit from cold stress. In addition, the participation of LeMAPK4 in cold-induced ethylene biosynthesis in tomato fruit was indicated. © 2013 Society of Chemical Industry

Nitric oxide (NO) is an important signal molecule involved in numerous plant responses to biotic and abiotic stress. C-repeat/dehydration-responsive element ( CRT / DRE )-binding factors (CBFs) play a prominent role in cold response regulation. To investigate the relationship between NO and the CBF pathway during cold stress, tomato ( Solanum lycopersicum cv. Lichun) fruit were treated with the NO donor (sodium nitroprusside, SNP) and nitric oxide synthase (NOS) inhibitor (N (omega)-nitro- l -arginine, l -NNA) before storage at 2 °C. Treatment with 0.02 mM SNP decreased the chilling injury (CI) index, whereas l -NNA treatment increased the appearance of chilling symptoms. NO content in fruit following SNP treatment was higher, while malondialdehyde (MDA) content and ion leakage were lower than that in water- and l -NNA-treated fruit. The relative expression of LeCBF1 , measured by q-RTPCR, in SNP-treated fruit was higher than that in l -NNA-treated and control fruit after cold storage for 0.5 and 4 h. These results suggest that SNP treatment protects tomatoes from cold injury by inducing NO accumulation and expression of LeCBF1 . NOS activity may play a role in NO accumulation associated with cold tolerance.

It has been known that methyl jasmonate (MeJA) interacts with ethylene to elicit resistance. In green mature tomato fruits (Lycopersicon esculentum cv. Lichun), 0.02mM MeJA increased the activity of 1-aminocyclopropane-1-carboxylate oxidase (ACO), and consequently influenced the last step of ethylene biosynthesis. Fruits treated with a combination of 0.02 MeJA and 0.02 α-aminoisobutyric acid (AIB, a competitive inhibitor of ACO) exhibited a lower ethylene production comparing to that by 0.02mM MeJA alone. The increased activities of defense enzymes and subsequent control of disease incidence caused by Botrytis cinerea with 0.2mM MeJA treatment was impaired by AIB as well. A close relationship (P

Nitric oxide (NO), an important signaling molecule in plants, is known to play crucial roles in the regulation of both defense responses and inducing resistance to fungal pathogens. To investigate NO synthesis during induced resistance and its mechanism, green mature tomatoes ( Lycopersicon esculentum cv. No.4 Zhongshu) were treated with the elicitor from Botrytis cinerea , considered an effective inducer of disease resistance, nitric oxide synthase (NOS) inhibitor N-nitro- l -arginine ( l -NNA) and nitric reductase (NR) inhibitor sodium azide (NaN 3 ). Our data indicated that reduced disease symptoms, and lesion size, in tomato fruit with B. cinerea were inhibited by 93.3 and 80.9% respectively three and four days after inoculation. NO biosynthesis was rapidly initiated one day after green mature tomatoes were treated with the elicitor and the peak NO content reached 3.43 μmol/g FW. The activities of NOS and defense enzymes, including phenylalanine ammonia-lyase (PAL), chitinase (CHI), β-1,3-glucanase (GLU), and polyphenoloxidase (PPO), were also increased by the elicitor treatment. Gene expression of pathogenesis-related protein 1(PR-1) increased within 2 h, and total phenols contents increased significantly within four days. A treatment of NOS inhibitor + elicitor increased disease incidence and lesion area and reduced the activities of defense enzymes compared with elicitor-treated fruit, indicating that elicitor-induced NO generation and disease resistance could be blocked by a NOS inhibitor. The NR inhibitor did not obviously influence the content of NO and disease resistance after incubation with the elicitor, and elicitor treatment did not induce NR activity. These results imply that NOS is a key enzyme regulating NO production in induced resistance.

The synthesis of high oil-absorption resins by suspended emulsion polymerization process for the first time with butyl methacrylate (BMA) and short-chain methyl methacrylate (MMA) as the monomers was studied. And the effects of different polymerization technological parameters, such as the comonomer, initiator, crosslinker, emulsifier, dispersant agent, and the agitation rate, on the oil absorbency of high oil-absorption resins were discussed in detail. The optimum polymerization conditions were obtained. With the increasing contents of these factors, the oil absorbency increased first, and then decreased. The highest oil absorbency to toluene was 17.6 g/g. The particle morphology of the high oil-absorption resins was observed by scanning electron microscopy (SEM). The resins were determined by FTIR spectrometry. Compared with the high oil-absorption resins prepared by suspension polymerization process and emulsion polymerization process, the high oil-absorption resins prepared by suspended emulsion polymerization process had the higher oil absorbency, faster oil-adsorbing rate, better oil-retention, and regeneration property. Copyright © 2010 John Wiley & Sons, Ltd.

Methyl jasmonate (MeJA), a major derivative of the plant hormone jasmonic acid, plays a critical role in inducing resistance to fungal pathogen. To study the endurance of MeJA-induced resistance and its cause, green mature tomatoes (Solanum esculentum cv. Lichun) were treated with 100 μM MeJA and nordihydroguaiaretic acid (NDGA, LOX inhibitor) at −35 kPa for 0.5 min and incubated at 25 ± 1 °C, 85–90% RH. Treatment with MeJA reduced disease symptoms in tomato fruit soon after being inoculated with Botrytis cinerea. Lesion size in MeJA-treated fruit was inhibited by 42.5%, 27.9% and 13.9% respectively (P < 0.05) in fruit inoculated 1, 3 and 6 d after treatments. At advanced stages (inoculation carried out 9 and 12 d after treatments), no inhibitory effect of MeJA were found. Ethylene biosynthesis was activated in the response of green mature tomatoes to methyl jasmonate with a rapid (1 d) and enhanced ethylene peak (0.9 ng kg−1 FW s−1). However the ethylene level was below that of the control from 6 d to 12 d. This rise was closely related with conversion of ACC to ethylene, especially a rise in ACO activity (6 h), which preceded an increase in ACS (12 h) after MeJA treatment. The development of ethylene biosynthesis was accompanied by a significant increase in LOX activity. Two significant O2− peaks (P < 0.05) were detected in MeJA-treated fruit during storage (6.18 μmol g−1 FW min−1 at 6 h and 5.68 μmol g−1 FW min−1 at 3 d). The correlations between LOX, and O2− and ACO activities were 0.75, 0.73 respectively (P < 0.05). These results indicate that MeJA-induced resistance against B. cinerea is durable, MeJA induces LOX and the superoxide radicals formed by LOX may activate ACO and ethylene biosynthesis.

Reverse atom transfer radical polymerization (RATRP) of methyl methacrylate (MMA) with azobisisobutyronitrile (AIBN)/FeCl3/lactic acid as initiating system was successfully carried out in N,N-dimethylformamide (DMF) for the first time. Kinetics as well as molecular weight distribution data indicated towards the controlled nature of polymerization. The polymerization not only showed the best control of molecular weight and its distribution but also provided a rather rapid reaction rate with the molar ratio of [MMA]:[AIBN]:[FeCl3]:[lactic acid] = 300 : 1 : 1 : 2. The polymerization rate increased with increasing the polymerization temperature. The apparent activation energy was calculated to be 47.90 kJ/mol. The rate of polymerization in DMF was faster than that in acetonitrile, cyclohexanone, toluene, and xylene. © 2009 Wiley Periodicals, Inc. J Appl Polym Sci, 2009

Porous acrylonitrile (AN)/itaconic acid (IA) copolymers were successfully prepared by suspended emulsion polymerization for the first time, with potassium peroxydisulfate (KPS) as an initiator, poly(vinyl alcohol) (PVA) as a dispersant agent, and Span80 as an emulsifier. The effects of the water/monomer mass ratio, agitation conditions, KPS concentration, PVA concentration, Span80 concentration,s and IA concentration on the average particle size and size distribution, particle morphology, and porosity of the AN/IA copolymers were investigated. The results show that the final AN/IA copolymers formed with agglomerates of primary particles had a porous structure, low particle density, and uniform particle size and did not agglomerate easily between the particles. The preparation conditions for the AN/IA copolymers were optimized as follows: (1) the water/monomer mass ratio was 0.3 : 1; (2) the concentrations of KPS, IA, PVA, and Span80 were 0.5, 12.4, 0.1, and 0.5 wt %, respectively, based on the weight of AN separately; (3) the agitation rate was 400 rpm; (4) the polymerization temperature was 70°C; and (5) the reaction time was 3 h. The size of the final AN/IA copolymer particles was in the range 200–400 μm. © 2008 Wiley Periodicals, Inc. J Appl Polym Sci, 2009

Highly crosslinked poly(styrene-co-divinylbenzene) microspheres with a 1.0–2.5-μm diameter were prepared by precipitation polymerization in neat acetonitrile. The effects of various polymerization parameters such as the monomer and initiator concentration, comonomer composition, and cosolvents on the properties of the resulting particles were studied. The particle diameter increased with the monomer concentration, whereas the particle size distribution remained almost constant. The resulting yield of the particles reached 41.1% when 6 wt % 2,2′-azobisisobutyronitrile (based on the total monomer) was used as the initiator. The styrene fraction in the monomer composition and toluene fraction in the solvent mixture also significantly affected the morphology and size of the resulting particles. © 2008 Wiley Periodicals, Inc. J Appl Polym Sci, 2009

BACKGROUND: Nitric oxide (NO) and hydrogen peroxide (H2O2) are signal molecules involved in the disease response of plants, and there is a close relationship between them. To investigate the interaction of NO and H2O2 during disease resistance response in postharvest fruits and vegetables, tomato (Lycopersicon esculentum cv. Lichun) fruits were treated after harvest by vacuum infiltration with NO donor and H2O2 scavenger. RESULTS: The resistance of tomato fruits to Rhizopus nigricans Ehrenb. invasion and the activities of defensive enzymes phenylalanine ammonia-lyase, chitinase and glutathione S-transferase were enhanced by NO. However, these effects of NO on resistance were weakened by H2O2 scavenger, which showed that H2O2 was required in NO-mediated disease resistance in harvested tomato. Meanwhile, the endogenous H2O2 level was dual-regulated by NO. During the earlier period of storage the H2O2 peak was advanced by NO, but during the later period of storage, when the concentration of H2O2 was relatively higher, H2O2 accumulation was delayed by NO. The activities of superoxide dismutase, ascorbate peroxidase and catalase were involved in this regulation. CONCLUSION: The results imply that application of exogenous NO can enhance the disease resistance of postharvest tomato; NO may interact with H2O2 and exert its effect by modulating the endogenous H2O2 level. This result is useful for the further investigation and application of NO in postharvest disease control. Copyright © 2008 Society of Chemical Industry

An experimental investigation with the combined use of visualization and measurement techniques was performed on flow pattern transitions and wall temperature distributions in the condensation of steam in silicon microchannels. Three sets of trapezoidal silicon microchannels, having hydraulic diameters of 53.0 µm, 77.5 µm and 128.5 µm, respectively, were tested under different flow and cooling conditions. It was found that during the transitions from the annular flow to the slug/bubbly flow, a peculiar flow pattern injection flow appeared in silicon microchannels. The location at which the injection flow occurred was dependent on the Reynolds number, condensation number and hydraulic diameter. With increase in the Reynolds number, or decrease in the condensation number and hydraulic diameter, the injection flow moved towards the channel outlet. Based on the experimental results, a dimensionless correlation for the location of injection flow in functions of the Reynolds number, condensation number and hydraulic diameter was proposed for the first time. This correlation can be used to determine the annular flow zone and the slug/bubbly flow zone, and further to determine the dominating condensation flow pattern in silicon microchannels. Wall temperature distributions were also explored in this paper. It was found that near the injection flow, wall temperatures have a rapid decrease in the flow direction, while upstream and downstream far away from the injection flow, wall temperatures decreased mildly. Thus, the location of injection flow can also be determined based on the wall temperature distributions. The results presented in this paper help us to better understand the condensation flow and heat transfer in silicon microchannels.

A hexa-substituted ethane thermal iniferter, diethyl-2,3-dicyano-2,3-di(p-tolyl) succinate (DCDTS), was firstly used as the initiator in the reverse atom transfer radical polymerization (RATRP) of acrylonitrile. FeCl3 coordinated by isophthalic acid (IA) was used as the catalyst in this system. The polymerization in N,N-dimethylformamide not only shows the best control of molecular weight and its distribution but also provides rather rapid reaction rate with the ratio of [AN] : [DCDTS] : [FeCl3] : [IA] at 500 : 1 : 2 : 4. The polymers obtained were end-functionalized by chlorine atom, and they were used as macroinitiators to proceed the chain extension polymerization in the presence of FeCl2/IA catalyst system via a conventional ATRP process and polyacrylonitrile obtained was with Mn = 39,260, PDI = 1.25. © 2007 Wiley Periodicals, Inc. J Appl Polym Sci 2007

Monodisperse crosslinked polystyrene microspheres were prepared by the dispersion copolymerization of styrene and divinylbenzene in a mixed solvent of ethanol and H2O. 2,2′-Azobisisobutyronitrile and poly(N-vinyl pyrrolidone) were used as the initiator and steric stabilizer, respectively. The crosslinker-postaddition method was adopted through a slow addition of a crosslinking agent into the dispersion system at a certain time after the beginning of the polymerization. The effects of the postaddition recipe, postaddition beginning time, postaddition velocity, and agitation rate on the particle size, size distribution, and morphology were discussed. The resulting polymer microspheres were characterized with scanning electron microscopy and laser particle analysis. Crosslinked polystyrene microspheres with a narrow size distribution and a 12.0% crosslinker level were obtained with a size of 1.0 μm through the crosslinker-postaddition method. © 2007 Wiley Periodicals, Inc. J Appl Polym Sci 2008

A well-defined organocobalt salen complex (salen)Co–CO2CH3 is used as a versatile photoinitiator for visible light-initiated living radical polymerization., The control of the polymerization of a wide range of monomers under mild conditions by a single catalyst remains a major challenge in polymer science. We report a versatile, well-defined organocobalt salen complex to control living radical polymerization of different categories of monomers, including acrylates, acrylamides and vinyl acetate, under visible light irradiation at ambient temperature. Both household light and sunlight were effectively applied in the synthesis of polymers with controlled molecular weights and narrow polydispersities. Narrowly dispersed block copolymers (M w/M n < 1.2) were obtained under various conditions. The structures of the polymers were analyzed by 1H NMR, 2D NMR, 13C NMR, GPC, MALDI-TOF-MS and isotopic labeling experiments, which showed that the ω and α ends of the polymer chains were capped with (salen)Co and –CO2CH3 segments, respectively, from the photoinitiator (salen)Co–CO2CH3. The ω end was easily functionalized through oxygen insertion followed by hydrolysis from 18O2 to –18OH. This robust system can proceed without any additives, and offers a versatile and green way to produce well-defined homo and block copolymers.

Catalytic hydrolysis of silanes mediated by chlorodicarbonylrhodium(I) dimer [RhCl(CO)2]2 to produce silanols and dihydrogen efficiently under mild conditions is reported. Second-order kinetics and activation parameters are determined by monitoring the rate of dihydrogen evolution. The mixing of [RhCl(CO)2]2 and HSiCl3 results in rapid formation of a rhodium silane σ complex.

Catalytic activation of an unstrained, unactivated Si-C(sp(3)) bond in water to form methane and silanol by electrophilic rhodium(III) porphyrin [(por)Rh(III)] in acidic aqueous solutions under visible light (λ ≥ 420 nm) has been developed. Activation of the Si-C(sp(3)) bond occurs through direct Si-C bond cleavage, with methyl group transfer to rhodium to give a porphyrin rhodium methyl complex. Photolysis of (por)Rh-CH(3) in water yields methyl radical and (por)Rh(II). Subsequently, (por)Rh(II) reacts with water rapidly to produce (por)Rh-H and (por)Rh-OH. (por)Rh-OH is then protonated to regenerate (por)Rh(III)-OH(2), and (por)Rh-H undergoes hydrogen atom abstraction by methyl radical to form the observed methane.

Acrylonitrile(AN) was successfully used to copolymerize with itaconic acid (IA) by suspension emulsion polymerization at 70 ˚C under N2 atmosphere for the first time. Porous AN/IA copolymers were prepared by using potassium peroxydisulphate (KPS) as initiator, poly(vinyl alcohol) (PVA) as dispersant and Span60 as emulsifier. Kinetics of suspension emulsion copolymerization of AN with IA were studied. Effects of various mass ratios of water/monomer, initiator concentrations, emulsifier concentrations and dispersant concentrations on AN/IA copolymerization rate and changes of particle size and size distribution with the extension of polymerization time were investigated. It was found that the copolymerization rate increased with water/monomer mass ratio and KPS concentration. Span60 concentration and PVA concentration had no obvious effect on the polymerization rate. Finally, a suspension emulsion polymerization mechanism for AN and IA was proposed.