Their structure, luminescence and magnetic properties were characterized by X-ray diffraction (XRD), scanning electronic microscope (SEM), transmission electron microscopy (TEM), photoluminescence (PL) spectra and vibrating sample magnetometer (VSM). The results indicated that the as-prepared multifunctional composites displayed a well-defined core-shell structure. The composites show spherical morphology with a size distribution of around 360 nm. Additionally, the composites exhibit high saturation magnetization (20.40 emu/g) and excellent luminescence properties. The inner Fe3O4 cores and the outer GdVO4:Dy3+ layers endow the composites with good responsive magnetic properties and strong fluorescent properties, which endow the nanoparticles with great potential applications in drug delivery, magnetic resonance imaging, and marking and separating of cells in vitro.In this study, multifunctional Fe In recent years, controlled drug delivery systems for modern drug therapy have been attracting increasing attention because they exhibit low toxicity, a wide therapeutic window, and ideal drug efficacy as compared to conventional drug delivery systems ,2. The m3O4 and lanthanide-doped nanomaterials. In these reports -2,5-diphenyltetrazolium bromide (MTT) viability histogram, shown in 3O4@SiO2@GdVO4:Dy3+ causes insignificant damage to the HeLa cells when the sample concentration increases to 200 μg·mL−1 for 24 h, and the cell viability remains at 90.38% even at the highest concentration, which indicates that Fe3O4@SiO2@GdVO4:Dy3+ nanoparticles are biocompatible.To evaluate the cytotoxicity of the Fe2O3 (99.9%) and Eu2O3 (99.9%) were purchased from Jinan Camolai Trading Company, Ferrous chloride hexahydrate (FeCl3·6H2O) (99%), tetraethyl orthosilicate , sodium acetate (NaAc), Citrate acid monohydrate were purchased from Beijing Chemicals Corporation. Nitric acid, ethanol, ethylene glycol (EG), and ammonia aqueous (25%) were purchased from Tianjin Chemicals Corporation. Deionized water obtained from the Milli-Q system  was used in all experiments. The magnetic Fe3O4 nanoparticles were prepared using a modified solvothermal reaction.All reagents are of analytical reagent grade and used without further purification. Gd3O4 nanoparticles were prepared according to a previously reported synthetic process [3·6H2O (1.3495 g) and NaAc (7.1926 g) were dissolved in EG solution (40 mL). Then PEG-10000 (1.0015 g) was added with vigorous stirring and the mixture was stirred for 30 min to form a homogeneous russet solution. The obtained solution was transferred to a Teflon-lined stainless-steel autoclave (50 mL capacity) and heated at 200 °C for 10 h. Subsequent cooling to room temperature yielded black magnetite particles, which were washed with ethanol and deionized water three times, respectively, and dried at 60 °C for 12 h.The magnetic Fe process . Typical3O4@SiO2 nanoparticles were prepared according to the modified by the Stöber method. In brief, 1.0 g of Fe3O4 nanoparticles were homogeneously dispersed in a mixture of 160 mL of ethanol, 40 mL of deionized water, and 3.0 mL of 28 wt % concentrated ammonia aqueous solution, followed by the addition of 3.0 mL of tetraethyl orthosilicate (TEOS). After vigorous stirring at 40 °C for 6 h, the obtained Fe3O4@SiO2 microspheres were separated with a magnet and washed repeatedly with ethanol and deionized water to remove nonmagnetic by products.Fe4:Dy3+ on the template Fe3O4@SiO2 was achieved according to the reported process with a doping concentration of Dy3+ of 0.5–4 mol % to Dy3+ in GdVO4:Dy3+. The typical procedure for synthesis is described as follows: stoichiometric amounts of Gd2O3, Dy2O3 and citric acid were dissolved in dilute nitric acid with heating followed by the addition of NH4VO3 in distilled water. Then PEG-10000 was added with a concentration of 0.05 g·mL−1. After stirring for 0.5 h, a homogenous sol was formed. Then the desired amount of Fe3O4@SiO2 nanoparticles was added into the gel, after further stirring for another 3 h, the resulting material was dried at 120 °C for 12 h to obtain the precursors. Then the precursors were calcined at 700 °C for another 4 h. The obtained nanoparticles were denoted as Fe3O4@SiO2@GdVO4:Dy3+ . In the experiment, the corresponding untreated cells were used as control. First, the HeLa cells were pre-incubated in a 96-well plate (about 3000 cells per well) for 24 h. Second, 2 mg of the Fe3O4@SiO2@GdVO4:Dy3+ nanoparticles were added into 10 mL of 0.01 M phosphate buffered saline  to form a stable orange solution. Third, the above solution at concentrations of 6.25, 12.5, 25, 50, 100 or 200 μg·mL−1 was added to the cells. Six parallel-group experiments were simultaneously conducted for each concentration. After 24 h, the viability of HeLa cells was examined by a MTT assay.Cell viabilities of FeThe purities of all the nanoparticles were checked by X-ray diffraction (XRD) measurements at room temperature using Cu Kα radiation (Kα = 1.54059 Å). The morphology and microscope structure of all the nanocomposites were characterized by a scanning electronic microscope  and transmission electron microscopy . The room temperature magnetic hysteresis (M-H) loops were measured using a superconducting quantum interference device vibrating sample magnetometry . Luminescence spectra were recorded on a FluoroMax-4 spectrophotometer . The specific surface area was determined by the Brunauer-Emmett-Teller (BET) method. The HeLa cells were assayed for viability by using a microplate reader .3O4@SiO2@GdVO4:Dy3+, with a core-shell structure from a combination of hydrothermal reaction and the sol-gel process. The as-prepared nanocomposites, combining the merits of the good magnetic response of the assembled Fe3O4@SiO2 microspheres and the fluorescence property of GdVO4:Dy3+, displayed high surface area and biocompatibility. Therefore, our study may provide new insight and useful information for the design of diverse, functional nanocomposites as drug carriers.In summary, we report a novel magnetic/luminescence multifunctional nanocomposite, Fe


In this study, we focused on the cardiac effects of T2, which have been poorly investigated so far, by using both in vitro and ex vivo models. As a comparison, the response to T3 and T4 was also determined. Rat cardiomyoblasts (H9c2 cells) were used to determine T2, T3, and T4 uptake by high-performance liquid chromatography–tandem mass spectrometry. In the same experimental model, MTT test, crystal violet staining, and glucose consumption were investigated, using T2 concentrations ranging from 0.1 to 10 µM. To assess cardiac functional effects, isolated working rat hearts were perfused with T2, T3, or T4 in Krebs-Ringer buffer, and the hemodynamic variables were recorded. T2 was taken up by cardiomyoblasts, and in cell lysate T2 levels increased slowly over time, reaching higher concentrations than in the incubation medium. T2 significantly decreased MTT staining at 0.5–10 µM concentration (P < 0.05). Crystal violet staining confirmed a reduction of cell viability only upon treatment with 10 µM T2, while equimolar T3 and T4 did not share this effect. Glucose consumption was also significantly affected as indicated by glucose uptake being increased by 24 or 35% in cells exposed to 0.1 or 1.0 µM T2 (P < 0.05 in both cases). On the contrary, T3 did not affect glucose consumption which, in turn, was significantly reduced by 1 and 10 µM T4 . In the isolated perfused rat heart, 10 µM T2 produced a slight and transient reduction in cardiac output, while T3 and T4 did not produce any hemodynamic effect. Our findings indicate that T2 is taken up by cardiomyoblasts, and at 0.1–1.0 µM concentration it can modulate cardiac energy metabolism by increasing glucose consumption. Some evidence of toxicity and a transient impairment of contractile performance are observed only at 10 µM concentration. These effects appear to be specific for T2, since they are not reproduced by T3 or T4.3,5-diiodo- Ryanodine was purchased from Abcam, UK. Unless otherwise specified, all reagents were obtained from Sigma-Aldrich . Solvents for HPLC–MS/MS measurements were HPLC-grade, and the other chemicals were reagent-grade.H9c2 (2–1) rat cardiomyoblast cell lines were obtained from American Type Culture Collection . , and wiIn vitro viability tests indicated that T2 produced only minor toxic effects upon a chronic (24 h) treatment. MTT staining was slightly but significantly reduced at concentrations exceeding 500 nM. Since the MTT test depends on mitochondrial function, we also used the crystal violet assay, finding out some evidence of T2 toxicity only at the highest concentration (10 µM). As reported . The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health .ex vivo experiments on heart. AS carried out mass spectrometry measurements. RZ revised the manuscript and performed statistical analysis. SG designed and supervised the experimental work and wrote the manuscript.GS, MN, and LL designed and carried out cell culture experiments. SF performed the The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed a potential conflict of interests.


We conducted a systematic study of the control of selective adsorption behaviour through the type of central metals in a series of analogous MOFs that show closed-open structural transformations. 2dab)[M2(ox)3] , having different central metals with analogous crystal structures. We found that a significant change in the selective adsorption behaviour for EtOH over MeCHO and MeCN is caused by the type of central metals, without changes in the crystal structures of all phases (except the Ni compound). A systematic study of adsorption measurements and structural analyses of the analogous MOFs reveals for the first time that the framework flexibility around the central metals of MOFs is truly related to the selective adsorption behaviour.Closed–open structural transformations in flexible metal–organic frameworks (MOFs) are of interest for potential applications such as separation, because of their complete selectivity for the adsorption of specific guest molecules. Here, we report the control of the adsorption behaviour in a series of flexible MOFs, (H Rational control or intentional modulation of the guest inclusion properties of porous metal–organic frameworks (MOFs) is one of the most important issues for controlling the functionality of these frameworks in applications such as gas storage,222(dobdc)  MOFs has been reported to show different adsorption behaviours for various gases.2(dobdc). We believe that a significant change in selective adsorption behaviour could be created in flexible MOFs that show closed–open structural transformations through using different types of central metals, as some research indicates that structural changes in flexible MOFs could be affected by the type of central metals.2+ and Mn2+ ions and have different structures in the guest-free condition has been reported.We have focused on controlling the selective adsorption behaviour of flexible MOFs that exhibit closed–open structural transformations by means of the difference in the type of central metals in a series of homologous frameworks. We believe that the type of central metals is an important parameter for controlling selective adsorption behaviour because it fine-tunes the energetics of the framework distortion during the adsorption/desorption process. Thus far, some MOFs that show no closed–open behaviour have been investigated for the ability to control adsorption properties through the type of central metals.2dab)[Zn2(ox)3]·nH2O , that shows a closed–open structural transformation during its adsorption process (3+) and acceptor (ox2–) sites in the interlayer space; therefore, it can selectively adsorb hydroxyl-functionalized guest molecules such as H2O, MeOH, and EtOH over other guests. This MOF was the first material to show complete adsorption selectivity for the large polar guest EtOH over the smaller polar aprotic guests MeCN and MeCHO.2dab)[M2(ox)3]·nH2O , having different central metals with almost analogous crystal structures. The guest-free anhydrate states, M, also have analogous structures with the exception of the Ni analogue. A systematic study of the adsorption properties of the analogous MOFs reveals that selective adsorption behaviour for EtOH over other guests in the MOF is significantly changed by the type of central metals.Here, we report a systematic study on the effect of the type of central metals on the selective adsorption behaviour of MOFs that show closed–open structural transformations. We employed an oxalate-bridged layered MOF 2·4H2O , oxalic acid dihydrate (H2(ox)·2H2O) , 1,4-diaminobutane (dab) , and distilled water  was heated in a 50 ml Teflon-lined bottle. The mixture was heated to 130 °C and was maintained at that temperature for 24 h. It was then slowly cooled to room temperature over 168 h. The reaction temperature was controlled using a programmable oven. The brown coloured crystals were collected by filtration . After washing the samples with distilled water, the samples were dried under air . Elemental analysis was performed. (%) calcd for C10H26N2O18Fe2: C 20.92, H 4.57, N 4.88; found: C 20.94, H 4.51, N 4.88.A mixture of Fe(CH3COO)2·4H2O , H2(ox)·2H2O , dab , and distilled water  was heated in a 50 ml Teflon-lined bottle. The temperature program for the hydrothermal synthesis was the same as that for nH2OFe·. A rose pink coloured precipitate was collected by filtration. After washing the samples with distilled water, the samples were dried under air . Elemental analysis was performed. (%) calcd for C10H18N2O14Co2: C 23.64, H 3.57, N 5.51; found: C 23.54, H 3.46, N 5.51.A mixture of Co(CH3COO)2·4H2O , H2(ox)·2H2O , dab , and distilled water  was heated in a 50 ml Teflon-lined bottle. The temperature program for the hydrothermal synthesis was the same as that for nH2OFe·. A yellow-green coloured precipitate was collected by filtration. After washing the samples with distilled water, the samples were dried under air . Elemental analysis was performed. (%) calcd for C10H18N2O14Ni2: C 23.66, H 3.57, N 5.52; found: C 23.56, H 3.38, N 5.56.A mixture of Ni(CHnH2OZn·.nH2OMg· described below.We previously reported the synthesis of 2(ox)·2H2O , dab , and distilled water  was heated in a 50 ml Teflon-lined bottle. The temperature program for the hydrothermal synthesis was the same as that for nH2OFe·. Colourless microcrystals were collected by filtration. After washing the samples with distilled water, the samples were dried under air . Elemental analysis was performed. (%) calcd for C10H14N2O12Mg2: C 29.82, H 3.50, N 6.95; found: C 30.08, H 3.53, N 6.98.A mixture of MgO , H2OFe·6H and 2OMg·6H were determined by SCXRD for the first time. The structures of 2OZn·6H and 2OZn·2H were previously determined and reported.λ = 0.71073 Å). The SCXRD measurements for 2OFe·6H and 2OMg·6H were performed using as-synthesised crystals that were immediately cooled to a low temperature (under N2 flow) after being placed on a capillary tube from the mother liquid. The crystal structures were solved using a direct method (SIR2002)F2 using the full-matrix least-squares methods with SHELXL-97.2OFe·6H, the hydrogen atoms were refined using isotropic thermal factors.The structures of λ = 1.54059 Å; Cu-Kα). Synchrotron XRPD measurements were obtained using the BL-8B beamline at the KEK Photon Factory (λ = 0.8265 Å) and the RIKEN Materials Science Beamline (BL44B2) at SPring-8 (λ = 0.7997 Å).2O , MeOH (100%), and EtOH (100%) conditions after drying at 80 °C under vacuum overnight. The structure of Zn·4MeOH was solved by Rietveld refinement and was previously reported.XRPD measurements were performed using a Bruker D8 ADVANCE (–1). The temperature range was from room temperature to 500 °C and the heating rate was 5 °C min–1.The thermal stability and the adsorbed hydrated phase were evaluated by thermogravimetric analysis (TGA). TG measurements were carried out with Bruker TG-DTA 2000SA under nitrogen gas flow (100 ml min2 (77 K), H2O, MeOH, MeCN, MeCHO (288 K), EtOH, Me2CO, i-PrOH, n-PrOH, and n-BuOH were measured at 298 K using a BELSORP18-PLUS and BELSORP-max . Samples were thoroughly dehydrated by heating at 80 °C overnight.Adsorption/desorption isotherms for N2OZn·6H and 2OMg·6H were hydrothermally synthesised by heating a mixture of metal oxide (ZnO or MgO), oxalic acid, 1,4-diaminobutane, and distilled water at 130 °C. In the case of 2OFe·6H, 2OCo·6H, and 2ONi·6H, metal acetates (M(CH3COO)2·4H2O ) were used for the reaction instead of metal oxides. Single crystals for X-ray crystallography were successfully obtained for 2OZn·6H, 2OMg·6H and 2OFe·6H. Crystals of 2OZn·2H were obtained by drying 2OZn·6H crystals under ambient conditions.Crystals of 2OFe·6H, 2OZn·6H and 2OMg·6H were successfully determined, and the crystallographic data are shown in 2OZn·6H.2OFe·6H and 2OMg·6H were solved using the same space group (P21/n) as that for 2OZn·6H. As shown in 2OFe·6H and 2OMg·6H also formed the typical honeycomb-shaped layer framework consisting of [M2(ox)3]2–, which incorporated H2dab2+ ions in the voids as counter cations. The guest water molecules were trapped in the space between the layers. 2– anions, and the ammonium groups of H2dab2+ are located in the interlayer space and interacted through hydrogen bonds. As is the case with 2OZn·6H, the guest water molecules were strongly trapped both by the hydrogen bond donor (–NH3+) and acceptor (O atoms on ox2–) sites of the host through three types of hydrogen bonds. One site was between water and the hydrogen bond donor sites of –NH3+; another site was between water and the hydrogen bond acceptor sites of ox2– ions; and the third site was between neighboring water molecules. Each water molecule formed two hydrogen bonds with both hydrogen bond donor and accepter sites. It should be noted that the guest arrangements and configuration of the hydrogen bonds in 2OFe·6H and 2OMg·6H were approximately the same as those in 2OZn·6H, meaning that the central metals were successfully changed to other elements without significant distortion of the crystal structures. Single crystals of nH2OCo· and nH2ONi· could not be obtained in this synthesis; however, as shown in 2OCo·6H phase that is isostructural with 2OFe·6H, 2OMg·6H and 2OZn·6H. Note that the nH2ONi· compound only showed the dihydrate XRPD pattern even under humidified conditions, indicating that there is no 2ONi·6H phase, as mentioned below.To determine the structures of these MOFs, single-crystal X-ray diffraction (SCXRD) measurements were performed. The crystal structures of nH2OM· , which showed three-step weight loss at RT, 100–120 °C and 300–350 °C. Considering the chemical compositions of the samples and the temperature regions of the weight losses, the weight losses around RT and 100–120 °C can be attributed to the desorption of included water guests. The mass loss at 100–120 °C corresponded to the desorption of two water molecules per formula unit, indicating the existence of the 2OM·2H dihydrate phase. According to the SCXRD measurement results, it was clear that the transformation from 2OM·6H to 2OM·2H easily occurred at around room temperature and that there are three different hydrated phases consisting of 2OM·6H, 2OM·2H and anhydrate M. Note that the Ni compound did not show any weight loss around room temperature, suggesting that it did not have a stoichiometric 2OM·6H phase but only 2OM·2H and M phases, which was consistent with the XRPD measurement. The weight losses at approximately 300–350 °C were attributed to the decomposition of ox2– ligands and H2dab2+, indicating that the framework of (H2dab)[M2(ox)3] can stably exist below 300 °C.To characterize the hydrated phases and the thermal stabilities of these samples, thermogravimetric analysis (TGA) was performed under nitrogen gas flow. Fig. S1 ESI shows TG2OZn·2H was successfully determined using SCXRD. As reported in the literature,2OZn·2H had horizontal 2-D layers, whereas the 2OZn·6H had distorted 2-D layers, indicating that there was a distortion process that occurred during the desorption process.The crystal structure of 2 (77 K), H2O, MeOH, EtOH, MeCN and MeCHO (288 K), Me2CO, i-PrOH, n-PrOH and n-BuOH (298 K). The samples were dehydrated by heating at 80 °C under vacuum overnight before the measurements were recorded. The fundamental parameters are shown in Table S3.To clarify the effect of the type of central metals on the selective adsorption behaviour, adsorption/desorption isotherms were measured using NP/P0 corresponded to two water molecules, which was attributed to the stoichiometric hydration of M to form 2OM·2H. This step confirmed that all of the samples had a dihydrate phase of 2OM·2H, as evidenced by the TGA results. Fe, Co, Zn, and Mg showed additional adsorption of four more water molecules at higher humidity (approximately 0.8 P/P0), which was attributed to the transformation from 2OM·2H to 2OM·6H. This result indicated that these samples have three stoichiometric phases: anhydrate, dihydrate, and hexahydrate. Only the Ni did not show any additional adsorption in the high humidity region and only had anhydrate and dihydrate phases. In the case of MeOH adsorption , except in the case of Ni. This work is the first systematic study demonstrating the control of selective adsorption behaviour through the type of central metals using flexible MOFs that show closed–open structural transformations. As discussed below, we believe that the difference in adsorption behaviour was derived from the covalent character of the Zn2+ and Co2+ ions which make the framework more flexible during the adsorption process.In contrast with MeOH adsorption, there was a significant difference in the EtOH adsorption behaviour. 2, MeCN, MeCHO, Me2CO, i-PrOH, n-PrOH and n-BuOH. Almost no adsorption of N2 (77 K) indicated that the anhydrate phases did not have any apparent microporosity, which confirmed that the adsorption processes for H2O, MeOH and EtOH are attributable to closed–open adsorption behaviour. As we previously reported, Zn has excellent hydroxyl group recognition properties, particularly, a non-size selective adsorption for polar protic guests (EtOH over MeCN and MeCHO).Co, Ni, and Zn did not show any adsorption for aprotic guests such as MeCN and MeCHO despite the fact that these molecules are smaller than EtOH, the Co and Ni also displayed hydroxyl group recognition. All of the samples did not adsorb guest molecules larger than EtOH , indicating that a size limit of included guest molecules exists.M), exposure to water (approx. 0.5 and 1 P/P0 for 2OM·2H and 2OM·6H), methanol (approx. 1 P/P0 for M·4MeOH) and ethanol (approx. 1 P/P0 for M·3EtOH). The samples were placed inside a sealed glass capillary, dehydrated by heating at 80 °C overnight and then exposed to the desired guests. The XRPD patterns under these conditions and the cell parameter refinement results by fitting using the Pawley or Le Bail methods are shown in Fig. S3–S11 and Tables S4–S7  could not be distinguished from Zn2+ and Co2+ ions (EtOH adsorption) by the order of the fundamental parameters, such as ionic radius  > Co2+ (0.75 Å) > Zn2+ (0.74 Å) > Mg2+ (0.72 Å)), average bond length of M–O (Fe2+ (2.121 Å in 2OM·6H) > Zn2+ (2.087 Å) > Mg2+ (2.075 Å)), and cell volume (Fe (448.9 Å3 per formula (see Table S43) > Mg (443.2 Å3) > Zn (442.5 Å3)). However, these ions can be distinguished by the order of the complex formation constants (β1 and β2) for the ox2– ligand  ≥ Zn2+  > Fe2+  > Mg2+ ),2– ligands. According to the value of ionic potentials (≡ ion charge divided by ionic radius) of these samples (Mg2+ (2.78) > Zn2+ (2.70) > Co2+ (2.67) > Fe2+ (2.56)), Mg2+ has a strongest electrostatic interaction to oxalate ions. However, Mg2+ has a lower complex formation constant for the ox2– ligands than Zn2+ and Co2+, indicating that there is a high contribution of covalent character of Zn2+ and Co2+ to the chemical bond with ox2– ligands. We believe that the covalent character of the Co2+ and Zn2+ ions tended to allow a slight deformation of the surrounding ox2– ions during the gate-open adsorption process, making Zn and Co more flexible than Mg and Fe. We also believe that the significant control of selective adsorption behaviour through the type of central metals in this compound was realised because of the existence of framework distortion in the honeycomb layer of [M2(ox)3]2–, as was the case for our compound. [M2(ox)3]n– sometimes forms undulating layered structures accompanied by framework distortion e.g., 2OM·2H).Fig. S9 Fig. S12.35 The X Fig. S11. The pat2dab)[M2(ox)3] that showed selective adsorption for hydroxyl-functionalized guests . Difference in the type of central metals significantly affects adsorption behaviour for EtOH because of the induced differences in the framework flexibility. There was a tendency for Fe and Mg to be less flexible than Zn and Co. We conducted a systematic study of the control of selective adsorption behaviour through the type of central metals in a series of analogous MOFs that show closed–open structural transformations. This study is an important example of the selective adsorption property of MOFs and provides a new opportunity to achieve significant control of selective adsorption behaviour using flexible MOFs.In conclusion, we demonstrated the control of selective adsorption behaviour through the type of central metals. We successfully synthesised isostructural frameworks of (HSupplementary informationClick here for additional data file.Crystal structure dataClick here for additional data file.


The resulting active core/active shell/inert shell nanoparticles are determined to be about 11 times brighter than that of well-investigated (NaYF4:Yb3+30%/Tm3+0.5%)/NaYF4 active core/inert shell nanoparticles when excited at ~980 nm. The strategy for enhanced upconversion in Yb3+/Tm3+-codoped NaYF4 nanoparticles through directed energy migration might have implications for other types of lanthanide-doped upconversion nanoparticles.The luminescence efficiency of lanthanide-doped upconversion nanoparticles is of particular importance for their embodiment in biophotonic and photonic applications. Here, we show that the upconversion luminescence of typically used NaYF Then, the solution was evaporated to dryness under the argon gas purge. Next, 2 mmol CF3COONa, 6 mL oleic acid , 10 mL octadecene , and 6 mL oleylamine  were added into the three-necked flask. The resulting solution was then heated at 120 °C with magnetic stirring for 30 min to remove water and oxygen. The solution was then heated to 300 °C at a rate of about 12 °C per minute under argon gas protection, and kept at this temperature under vigorous stirring for about 0.5 h. A needle was used to let the argon gas out during the synthesis. The mixture was cooled to room temperature and precipitated by ethanol in an ultrasonic bath and collected by centrifugation at 7000 rpm for 5 min. The precipitate was then washed with ethanol for several times, and the nanoparticles were dispersed in 10 mL of hexane for further characterizations.NaYFs method . All che4: Yb3+30%/Tm3+0.5%)/NaYbF4 active core/active shell nanoparticle were synthesized following a similar preparation procedure as the core. A 0.5 mmol amount of Yb2O3 dissolved in 10 mL 50% concentrated trifluoroacetic acid at 95 °C in a three-necked 100 mL flask. Then, the solution was evaporated to dryness under the argon gas purge. Next, 1 mmol CF3COONa, 0.5 mmol NaYF4: Yb3+30%/Tm3+0.5% core, 10 mL oleic acid, and 10 mL octadecene were added into the three-necked flask. The resulting solution was heated at heated at 120 °C with magnetic stirring for 30 min to remove water and oxygen. The solution was then heated to 300 °C at a rate of about 12 °C per minute under argon gas protection, and kept at this temperature, under vigorous stirring, for about 0.5 h. The mixture was cooled to room temperature and precipitated by ethanol in an ultrasonic bath and collected by centrifugation at 7000 rpm for 5 min. The precipitate was then washed with ethanol several times, and the nanocrystals were dispersed in 10 mL of hexane for further characterizations.The (NaYF4: Yb3+30%/Tm3+0.5%)/NaYF4 active core/inert shell nanoparticles were synthesized following a similar preparation procedure as described in 2O3 was dissolved in 10 mL 50% concentrated trifluoroacetic acid at 95 °C in a three-necked 100 mL flask. Then, the solutions were evaporated to dryness under the argon gas purge. Next, 1 mmol CF3COONa, 0.5 mmol NaYF4:Yb3+30%/Tm3+0.5% core, 10 mL oleic acid, and 10 mL octadecene were added into the three-necked flask. The resulting solution was then heated at heated at 120 °C with magnetic stirring for 30 min to remove water and oxygen. The solution was then heated to 300 °C at a rate of about 12 °C per minute under argon gas protection, and kept at this temperature under vigorous stirring for about 0.5 h. The mixture was cooled to room temperature and precipitated by acetone in an ultrasonic bath and collected by centrifugation at 7000 rpm for 5 min. The precipitate was then washed with ethanol for several times, and the nanocrystals were dispersed in 10 mL of hexane for further characterizations.The (NaYF4: Yb3+30%/Tm3+0.5%)/NaYbF4/NaYF4 active core/active shell/inert shell nanoparticles were synthesized following a similar procedure as the core. A 0.25 mmol amount of Y2O3 dissolved in 50% concentrated trifluoroacetic acid at 95 °C in a three-necked 100 mL flask. Then, the solutions were evaporated to dryness under the argon gas purge. Next, 0.5 mmol CF3COONa, 0.25 mmol (NaYF4: Yb3+30%/Tm3+0.5%)/NaYbF4 active core/active shell nanoparticles, 5 mL oleic acid, and 5 mL octadecene were added into the three-necked flask. The resulting solution was then heated at heated at 120 °C with magnetic stirring for 30 min to remove water and oxygen. The solution was then heated to 300 °C at a rate of about 12 °C per minute under argon gas protection, and kept at this temperature under vigorous stirring for about 0.5 h. The mixture was cooled to room temperature and precipitated by ethanol in an ultrasonic bath and collected by centrifugation at 7000 rpm for 5 min. The precipitate was then washed with ethanol for several times, and the nanocrystals were dispersed in 10 mL of hexane for further characterizations.The /NaYbF4, the active core/inert shell (NaYF4: Yb3+30%/Tm3+0.5%)/NaYF4, the active core/active shell/inert shell (NaYF4: Yb3+30%/Tm3+0.5%)/NaYbF4/NaYF4 were recorded at the Infinium oscilloscope  coupled to the photomultiplied tube (PMT) of the Fluorolog-3.11 Jobin Yvon spectrofluorimeter, using excitation at 975 nm from laser diode (Q-Photonics) operating in pulsed mode.The size and morphology of UCNPs were characterized by transmission electron microscopy (TEM) using a JEM-2010 microscope  at an acceleration voltage of 200 KV. The powder X-ray diffraction (XRD) patterns were recorded by a Siemens D500 diffractometer using Cu Kα radiation (λ = 0.15418 nm). The 2θ angle of the XRD spectra was recorded at a scanning rate of 5°/min. UC luminescence spectra were recorded using a Fluorolog-3.11 Jobin Yvon spectrofluorometer  with a slit width defining spectral resolution of 2 nm. The luminescence was excited at 975 nm using a fiber-coupled laser diode (Q-Photonics). All UC luminescence spectra have been corrected for the spectral sensitivity of the system. Decays of luminescence at 802 nm for UCNPs of the core NaYF4: Yb3+30%/Tm3+0.5% UCNPs has been enhanced by ~240 times through utilization of the active core/active shell/inert shell (NaYF4: Yb3+30%/Tm3+0.5%)/NaYbF4/NaYF4 structure. Moreover, the UC luminescence of the active core/active shell/inert shell (NaYF4: Yb3+30%/Tm3+0.5%)/NaYbF4/NaYF4 UCNPs is determined to be about 11 times higher than that of well-investigated (NaYF4: Yb3+30%/Tm3+0.5%)/NaYF4 active core/inert shell UCNPs. Mechanistic investigation reveals that the significant UC enhancement in the active core/active shell/inert shell UCNPs arises from directed energy migration within the second active shell layer solely to the surface of the core nanoparticle. The guidance of energy migration is provided by the outmost inert shell which blocks the way of energy migration to surface quenching sites. The enhancement UC strategy demonstrated here in Yb3+/Tm3+-doped NaYF4 nanoparticles has implications to be extended to other type of lanthanide-doped UCNPs.The intensity of UC luminescence of NaYF


Enterobacter cloacae strains CAPREx E7 and CAPREx E2-2 were isolated from Ghanaian yams at a London market. The draft genome sequences indicate that the two strains are similar, with genomes of 5,042,838 and 5,039,930 bp and 56.19% and 55.05% G+C content, respectively. Both strains encoded three different β-lactamases, including one of the AmpC family. Enterobacter cloacae is a Gram-negative rod-shaped member of the family Enterobacteriaceae. E. cloacae strains are often associated with human infections, and many strains of E. cloacae carry β-lactamases or carbapenemases that confer antibiotic resistance  and MWMD00000000 (CAPREx E7).The draft genomes of these two strains have been deposited in GenBank under accession no.


DDA1 (DET1 and DDB1 associated 1) was first discovered and registered in the GenBank database by our colleagues. DDA1, an evolutionarily conserved gene, might have significant functions. Recent reports have demonstrated that DDA1 is linked to the ubiquitin–proteasome pathway and facilitates the degradation of target proteins. However, the function of DDA1 in lung cancer was previously unknown. This study aimed to investigate whether DDA1 contributes to tumorigenesis and progression of lung cancer. We found that the expression of DDA1 in normal lung cells and tissue was significantly lower than that in lung cancer and was associated with poor prognosis. DDA1 overexpression promoted proliferation of lung tumour cells and facilitated cell cycle progression in vitro and subcutaneous xenograft tumour progression in vivo. Mechanistically, this was associated with the regulation of S phase and cyclins including cyclin D1/D3/E1. These results indicate that DDA1 promotes lung cancer progression, potentially through promoting cyclins and cell cycle progression. Therefore, DDA1 may be a potential novel target for lung cancer treatment, and a biomarker for tumour prognosis.Lung cancer is globally widespread and associated with high morbidity and mortality.  DDA1 was first discovered by our colleagues through the serological analysis of recombinant cDNA expression libraries (SEREX) technique as a gene encoding a 1086‐bp cDNA and a 309‐bp open reading frame Lung cancer is the leading cause of cancer‐related mortality in the United States, accounting for more deaths than breast, prostate, colon and pancreatic cancers combined Loss of growth control is a hallmark of all cancers in vitro through G1/S transition and S‐phase acceleration and regulation of cyclins. In addition, inhibition of DDA1 was shown to suppress tumorigenesis in vivo in a subcutaneous xenograft mouse model. Taken together, these results indicate that DDA1 promotes the progression of lung cancer by regulating the cell cycle, especially S phase, and cyclins such as cyclin D1/D3. DDA1 could be a powerful indicator of tumour prognosis in patients with lung cancer.These results motivated us to investigate whether DDA1 participates in lung cancer tumorigenesis and tumour progression through the regulation of cyclins and cell cycle progression and thus could serve as a prognostic biomarker for lung cancer. Here, we demonstrated that DDA1 levels were increased in both lung adenocarcinoma and lung squamous cell carcinoma and that high expression of DDA1 was associated with poor prognosis. DDA1 promoted lung cancer cell proliferation, increased cell cycle progression 2. Cells were transfected by X‐tremeGENE HP DNA Transfection Reagent . Plasmids pcDNA3.1(+) (Mock), pcDNA3.1(+)‐DDA1 (DDA1), pRNA‐U6.1‐CTL (shMock) and pRNA‐U6.1‐shDDA1 (shDDA1) were purchased from GenScript . All shRNA sequences are shown in Table MRC‐5, NCI‐H292, NCI‐H526, 95‐D, NCI‐H441, NCI‐H358, A549, NCI‐H1299, Calu‐1, NCI‐H460, SPC‐A1, NCI‐H1975, NCI‐H69, NCI‐H446, NCI‐H1993 and NCI‐H2228 cell lines were obtained from the American Type Culture Collection . Cells were cultured in RPMI‐1640  with 10% FBS  and 1% penicillin and streptomycin with humidity at 37°C and 5% COn = 100; T047, n = 18). Tissue microarrays with survival data were purchased from Shanghai Outdo Biotech CO. LTD. . The institutional review board approved the use of de‐identified samples; informed consent was obtained from all patients. A total of 418 tissues were analysed for DDA1 expression by IHC according to the manufacturer's recommendations . IHC scores were calculated as previously described Tissue microarrays containing FFPE  samples of lung cancer, adjacent tissue and normal lung tissues were purchased from US Biomax, Inc.  was used, and absorbance was measures at 450 nm for each well at different time‐points using a microplate reader . For colony formation assays, transfected cells were plated at 500–1000 cells per well and three wells for each group into six‐well plates and cultured for approximately 14 days, followed by crystal violet staining.For cell growth assays, transfected cells were seeded at 2 × 10Cells were collected and fixed with 70% ethanol. The cell pellet was then treated with 50 μg/ml propidium iodide  containing 0.1 mg/ml RNase A (Sigma‐Aldrich) and 0.1% Triton X (Sigma‐Aldrich), which was followed by flow cytometric analysis . Data were analysed using FlowJo 9.1 software .For thymidine and nocodazole blocking and release experiments, cells were treated with 2 mM thymidine (Sigma‐Aldrich) for 24 hrs and released for 9 hrs, followed by 200 ng/ml nocodazole (Sigma‐Aldrich) for 12–18 hrs. For cell cycle analyses, processing was performed as described previously herein.7 cells in 100 μl of RPMI‐1640) were injected subcutaneously into the flanks of female BLAB/c nude mice , China). Tumour size was determined by collecting length and width with a sliding caliper every 3 days, and calculating the tumour volume (mm3) as length × (width)2 × 0.52. When mice were killed, tumours from each animal were collected, weighed and used for histopathological studies and Western blotting. Procedures involving animals conformed to the guidelines of the Institutional Animal Care and Use Committee of West China Hospital, Sichuan University.Lung cancer cells A549/LV‐mock, A549/LV‐DDA, H1299/LV‐shMock and H1299/LV‐shDDA1  was used to analyse all data. P < 0.05 was regarded as statistically significant.A chi‐square test or Fisher's exact test was used to compare the differences in categorical variables. A Student's As a novel gene, the DDA1 expression profile had not been documented in mouse tissues and organs, previously. We analysed DDA1 protein and mRNA by Western blot and RT‐PCR, respectively, in 14 types of organs or tissues of C57BL/6j mouse under normal physiological conditions. We found that DDA1 expression was relatively lower in the lung and heart, while it was relatively higher in the spleen and thymus  and H441 (Mock: 1.021 ± 0.005 versus DDA1: 1.253 ± 0.022) cells  and H292 (shMock: 0.751 ± 0.012 versus shDDA1: 0.520 ± 0.007) cells  and H441 (Mock: 173.7 ± 10.27 versus DDA1: 268.3 ± 14.40) cells  and H292 (shMock: 447.7 ± 20.51 versus shDDA1: 314.0 ± 11.02) cells was hindered following inhibition of DDA1 expression     Fig. I and J. 01) Fig. K and tumin vitro, we assessed the cell proliferation rate by CFDA‐SE (5‐(and‐6)‐carboxyfluorescein diacetate, succinimidyl ester) staining and flow cytometry. After staining with CFDA‐SE for 4 days, the fluorescence intensity of A549 cells overexpressing DDA1 was lower than that of control cells, indicating that DDA1 increased the frequency of cell division  and an increased percentage of S‐phase (Mock: 37.00 ± 0.46% versus DDA1: 43.20 ± 0.32%) cells  and a decreased proportion of S‐phase (shMock: 34.03 ± 0.67% versus shDDA1: 25.20 ± 0.70%) cells    or decreased (shMock: 34.1 ± 1.2% versus shDDA1: 23.8 ± 1.0%) the proportion of BrdU‐positive (S‐phase) cells  Figs  in A549 2%) Figs . Furtherversus DDA1: 3.50 ± 0.08%) or decreased (shMock: 2.31 ± 0.07% versus shDDA1: 1.41 ± 0.08%) mitotic entry with overexpression or silencing of DDA1  xenografts from nude mice , cyclin D1 (58.20 ± 4.22% versus 12.10 ± 2.48%), cyclin D3 (47.20 ± 4.50% versus 10.10 ± 1.91%), cyclin E1 (41.30 ± 4.22% versus 11.20 ± 3.12%) and cyclin B1 (32.10 ± 3.12% versus 9.40 ± 1.96%) levels than control tissues  of DDA1 expression using a commercially available tissue microarray (TMA) containing 118 cases of lung cancer, adjacent tissue and normal tissue. Negative and positive staining of DDA1 in the epithelial cells was observed in the normal lung tissue, whereas DDA1 expression was high in lung cancer and the adjacent region, in most cases    Figs B and S5.05) Fig. C.P = 0.002) and AJCC stage (P = 0.016) , tumour size (P = 0.012), positive LN number (P = 0.022) and AJCC stage (P < 0.001) were found to have an adverse impact on overall survival in patients with lung cancer (Table P = 0.005), tumour size (P = 0.012), positive LN number (P < 0.001) and AJCC stage (P < 0.001) were significant predictors of overall survival  A549 cells were transfected and cultured for 2 days followed by PI staining and flow cytometry for cell cycle analysis. The ratio of G0/G1 phase was decreased and the proportion of cells in S and G2/M phases was significantly increased after overexpression of DDA1. (B) H1299 cells were transfected and cultured for 2 days followed by PI staining and flow cytometry for cell cycle. The ratio of G0/G1 phase was increased and proportion of cells in S and G2/M phases was significantly decreased after inhibition of DDA1Figure S3 (A) A549 cells were transfected and cultured for 24 hrs followed by synchronization to G2/M phase by thymidine and nocodazole. The cells were released from blocking for indicated times and analyzed by PI staining and flow cytometry. The proportion of S‐phase cells was significantly increased after 6 hrs (B) H1299 cells were transfected and treated as in (A). Then cells were released from blocking for indicated times and analyzed by PI staining and flow cytometry. The percentage of S‐phase cells was decreased significantly after 10 hrsFigure S4 DDA1 is overexpressed in lung cancer tissue. 8 pairs of tumor (T) and normal (N) tissue of lung cancer patients were assessed by western blot and DDA1 level in all these tumor tissues was higher than that of normal tissuesFigure S5 Representative IHC score of TMA tissue sectionsTable S1 shRNA sequence of DDA1Table S2 Primers used for qPCRClick here for additional data file.


H)-one (BZ3) and 3-((4-(dimethylamino)benzylidene)amino)-2-methylquinazolin-4(3H)-one (BZ4), were employed as highly efficient inhibitors of mild steel corrosion by corrosive acid.Relatively inexpensive, stable Schiff bases, namely 3-((4-hydroxybenzylidene)amino)-2-methylquinazolin-4(3The inhibition efficiencies were estimated based on weight loss method. Moreover, scanning electron microscopy was used to investigate the inhibition mechanism. The synthesized Schiff bases were characterized by Fourier transform infrared spectroscopy, nuclear magnetic resonance spectroscopy and micro-elemental analysis. The inhibition efficiency depends on three factors: the amount of nitrogen in the inhibitor, the inhibitor concentration and the inhibitor molecular weight.N,N-dimethylamino substituents on the inhibition efficiency, providing insight for designing new molecular structures that exhibit enhanced inhibition efficiencies.Inhibition efficiencies of 96 and 92% were achieved with BZ4 and BZ3, respectively, at the maximum tested concentration. Density functional theory calculations of BZ3 and BZ4 were performed to compare the effects of hydroxyl and  H)-one (BZ3) and 3-((4-(dimethylamino)benzylidene)amino)-2-methylquinazolin-4(3H)-one (BZ4) were synthesized. Their molecular structures were determined by elemental analysis; carbon, hydrogen and nitrogen (mass fractions of CHN) analysis, Fourier transform infrared FTIR spectroscopy and nuclear magnetic resonance (NMR) spectroscopy. The abilities of these molecules to inhibit mild steel corrosion in an acidic solution were determined by the weight loss method and scanning electron microscopy (SEM). To elucidate the inhibition mechanism and the relationship between the structure and inhibition efficiency of the inhibitor, quantum chemical calculations of BZ3 and BZ4 were performed.Anti-corrosion coatings are generally employed to inhibit the average of corrosion and increase longevity of the mild steel. A broad range of organic adsorption inhibitors presently applied in the corrosion domain are expensive , 2. ElecAll chemical compounds were purchased from Sigma-Aldrich/Malaysia. Fourier transform infrared (FTIR) spectra were recorded on a Shimadzu FTIR-8300 spectrometer. Elemental analyses were performed using a Carlo Erba 5500 elemental analysis; carbon, hydrogen and nitrogen (CHN). Nuclear magnetic resonance spectra were obtained using a Bruker Spectrospin instrument equipped with 300 MHz UltraShield magnets. DMSO-d6 and TMS were used as the solvent and internal standard, respectively.H)-one (0.005 mol), the appropriate carbonyl compound (0.005 mol) and a few drops of acetic acid were refluxed for 8 h. After cooling, the mixture was filtered, and the obtained solid was subsequently washed and recrystallized from hot ethanol. BZ3: yield 72%, mp 204–206 °C. FTIR: 3189 , 1704.3 (C=O), 1609.0 (C=N). 1H NMR: 2.37 , 6.84–7.01 , 5.32 , 9.33 . Elemental analysis (CHN): C 69.11% (68.81%), H 4.91% (4.69%), N 14.82 (15.05). BZ4: yield 68%, mp 191–193 °C. FTIR: 3047.4 (aromatic C–H), 1699.6 (C=O), 1611.3 (C=N). 1H NMR: 2.410 , 7.01–7.32 , 8.99 . Elemental analysis (CHN): C 70.90% (70.57%), H 6.03% (5.92%), N 18.78 (18.29%).An ethanolic solution of 3-amino-2-methylquinazolin-4-one with 4-hydroxybenzaldehyde and N,N-dimethyl-4-aminobenzaldehyde, respectively. The molecular weights of BZ3 and BZ4 were estimated to be 279 and 306, respectively, from the chemical formulas  and were confirmed by spectroscopic techniques. No hydrazide absorption bands were observed in the BZ3 and BZ4 FTIR spectra. The BZ3 1H NMR (nuclear magnetic resonance) spectrum exhibited singlets at δ 5.32 ppm, due to the OH proton, and δ 2.37 ppm (3H), due to the methyl group. In the BZ4 1H NMR spectrum, only one singlet was observed at δ 2.410 (3H) due to the methyl group. The Schiff bases were synthesized from 3-amino-2-methylquinazolin-4(3H)-one according to the procedure illustrated in Scheme The Schiff bases BZ3 and BZ4 were readily synthesized in excellent yields by refluxing 3-amino-2-methylquinazolin-4 for  and 303 K for mild steel in corrosive media. The BZ3 and BZ4 results, which are shown in Figs. To determine the effect of the temperature on the inhibition efficiency, corrosion experiments were performed in the absence or presence of BZ4 at various temperatures . The inhibition performance was enhanced by increasing the BZ4 concentration and decreasing the temperature. Figure The efficiencies of the investigated inhibitors BZ3 or BZ4 could rely on charges or molecular weights, in addition to the nature of bonds of the metal and its capability to produce complexes. Figure 2+ electron configuration is [Ar]3d6. The 3d orbitals mix with the unoccupied 4s and 4p orbitals to form sp3 or d2sp3 hybrid orbitals that might be suitably oriented toward the nitrogen or oxygen non-bonding electron pairs in the inhibitors. When these Fe and inhibitor orbitals overlap, tetrahedral, square planar or octahedral complexes in which the metal has a filled valence shell are formed. The inhibition mechanism can also be explained in terms of crystal field theory (CFT) or molecular orbital theory (MOT). When the inhibitor molecules complex to the metal atoms, coordination bonds form via electron transfer from the inhibitor nitrogen atoms to the metal d orbitals.The inhibition mechanism of the tested inhibitors can be explained by valence bond theory (VBT). The FeThe mild steel surface was analyzed by SEM after immersion in 1.0 M HCl with and without 0.5 g/L BZ4 for 3 h at 30 °C, as shown in Fig. H)-one (BZ3) with a hydroxyl (–OH) group and 3-((4-(dimethylamino)benzylidene)amino)-2-methylquinazolin-4(3H)-one (BZ4) with an N,N-dimethylamino (–NMe2) group, were studied by DFT. Two additional isomer models of both BZ3 and BZ4 were also investigated [To elucidate the significant electronic effects of the substituents, the two inhibitors with strongly electron-donating groups, namely 3-((4-hydroxybenzylidene)amino)-2-methylquinazolin-4(3stigated .The hydroxyl group on the benzene ring in BZ3 is in the C-4 position but could be moved to the C-2 (BZ3a) and C-3 (BZ3b). For all three positions, the contribution of the substituent to both the HOMO and LUMO was similar with only small variations, as shown in Fig. The ionization potential (I) and electron affinity (A) were calculated according to Koopmans’ theorem , 39 as fI = − EHOMO; A = − ELUMOIadd% is the percent change in the ionization potential of model x-BZ3 relative to that of BZ3, and Ieadd% and Ietheory% are the corresponding additional and theoretical inhibition efficiencies, respectively.The method of Al-Amiery et al. , 39 was meta position (BZ3b) led to a decrease in the inhibition efficiency to 77.81%, whereas moving it to the ortho position (BZ3a) resulted in an increase in the inhibition efficiency to 96.11%. A comparison of the BZ3 and BZ3a inhibition efficiencies (96.11% vs. 92%) reveals that this change in the substituent position clearly enhanced the inhibition efficiency.These results demonstrate that moving the hydroxyl group to the N,N-dimethylamine group on the benzene ring in BZ4 is in the C-4 position but could be moved to the C-2 (BZ4a) and C-3 (BZ4b) positions. For all three positions, the contribution of the substituent to both the HOMO and LUMO was similar with only small variations, as shown in Fig. The The ionization potential (I) and electron affinity (A) were calculated according to Koopmans’ theorem  as folloI = − EHOMO; A = − ELUMOThe inhibition efficiencies of the BZ4 isomers calculated using Eqs. N,N-dimethylamino substituent to the meta position (BZ4b) led to a decrease in the inhibition efficiency to 85.27%, whereas moving it to the ortho position (BZ4a) resulted in an increase in the inhibition efficiency to 94.98%. This result along with that for BZ4 (96%) reveals that an excellent inhibition efficiency could be achieved with BZ4 isomers.These results demonstrate that moving the meta directors. Also, the groups that have unoccupied pair of electrons, like the amino group (BZ4) or hydroxyl group (BZ3), are strong active and ortho (BZa)/para-directors (BZ) thus efficient groups donate the unoccupied electrons to the pi system, making a negative charge on ortho (position-2) and para (position-4)positions. These positions have the maximum activities toward electron-poor electrophile. The highest electron density have been located on ortho/para positions, although. An important point; steric hindrance as in compound BZ4 that have 2-methyl groups on nitrogen atom  decrease the reactivity. The final result of the electrophilic aromatic substitution might thus be hard to predict, and it is usually only established by doing the reaction and determining the ratio of ortho versus para substitution.Groups which were withdrawing electron by resonance effect will decrease density of electrons specifically at positions 2, 4 and 6, leaving position 3 and position 5 as the ones with relatively higher efficiency, thus these kinds of groups were (position-3) ortho-position) for the compound BZ3a and no steric hindrance.Finally, from Table H)-one (BZ3) and 3-((4-(dimethylamino)benzylidene)amino)-2-methylquinazolin-4(3H)-one (BZ4), exhibited excellent corrosion inhibition performances, and maximum inhibition efficiencies of 96 and 92% were observed for BZ4 and BZ3, respectively, at an inhibitor concentration of 5 mM. The inhibition efficiency increased with increasing inhibitor concentration, whereas it decreased with increasing temperature. The SEM images show that BZ4 might form a protective film on the mild steel surface.Mild steel corrosion inhibitors were synthesized, and their structures were fully characterized by spectroscopic techniques. Their abilities to inhibit mild steel corrosion in a 1.0 M HCl solution at 303, 313, 323 and 333 K were subsequently studied. The inhibitors, namely 3-((4-hydroxybenzylidene)amino)-2-methylquinazolin-4 calculations of BZ3 and BZ4 isomers revealed that a substituent in the


Dengue viruses (DENVs) are significant mosquito-transmitted pathogens that cause widespread infection and can lead to severe infection and complications. Here we further characterize a novel and robust DENV serotype 1 (DENV1) infectious clone system that can be used to support basic and applied research. We demonstrate how the system can be used to probe the antigenic relationships between strains by creating viable recombinant viruses that display or lack major antibody epitopes. The DENV1 clone system and recombinant viruses can be used to analyze existing vaccine immune responses and inform second-generation bivalent vaccine designs. Despite the clinical relevance, dengue virus (DENV) research has been hampered by the absence of robust reverse genetic systems to manipulate the viral serotypes for propagation and generation of mutant viruses. In this article, we describe application of an infectious clone system for DENV serotype 1 (DENV1). Similar to previous clones in both flaviviruses and coronaviruses, the approach constructs a panel of contiguous cDNAs that span the DENV genome and can be systematically and directionally assembled to produce viable, full-length viruses. Comparison of the virus derived from the infectious clone with the original viral isolate reveals identical sequence, comparable endpoint titers, and similar focus staining. Both focus-forming assays and percent infection by flow cytometry revealed overlapping replication levels in two different cell types. Moreover, serotype-specific monoclonal antibodies (MAbs) bound similarly to infectious clone and the natural isolate. Using the clone, we were able to insert a DENV4 type-specific epitope recognized by primate MAb 5H2 into envelope (E) protein domain I (EDI) of DENV1 and recover a viable chimeric recombinant virus. The recombinant DENV1 virus was recognized and neutralized by the DENV4 type-specific 5H2 MAb. The introduction of the 5H2 epitope ablated two epitopes on DENV1 EDI recognized by human MAbs (1F4 and 14C10) that strongly neutralize DENV1. Together, the work demonstrates the utility of the infectious clone and provides a resource to rapidly manipulate the DENV1 serotype for generation of recombinant and mutant viruses.IMPORTANCE Dengue viruses (DENVs) are significant mosquito-transmitted pathogens that cause widespread infection and can lead to severe infection and complications. Here we further characterize a novel and robust DENV serotype 1 (DENV1) infectious clone system that can be used to support basic and applied research. We demonstrate how the system can be used to probe the antigenic relationships between strains by creating viable recombinant viruses that display or lack major antibody epitopes. The DENV1 clone system and recombinant viruses can be used to analyze existing vaccine immune responses and inform second-generation bivalent vaccine designs. Dengue viruses (DENVs) represent a significant threat to human health and the global economy, causing widespread and frequent epidemics , 2. TranTo study aspects of infection and pathogenesis, a number of reverse genetic systems have been generated for members of the flavivirus family, which includes DENV , 7. Thes10–Yet, while current systems have been useful, several obstacles remain for DENV reverse genetic systems . Flavivi13–In this study, we extend our characterization of a dengue virus serotype 1 (DENV1) infectious clone  by compaThe recombinant DENV1 infectious clone (rDENV1ic) was synthesized as a panel of four continuous cDNA segments that span the entire genome  19). Ba. Ba19). RNA isolated from infected cells was verified relative to the parental wild-type strain using genome length analysis and finding no differences in the consensus amino acid sequences. Similarly, endpoint titers from C6/36 cells  found noSome of the previously described reverse genetic systems for DENVs have been hampered by replication attenuation relative to the wild-type strain despite identical consensus sequences . To deteAntibody binding and neutralization are critical issues in DENV pathogenesis in humans; as such, the infectious clone and parental WT strain must display the same surface architecture and epitopes. To compare the display of antibody epitopes, enzyme-linked immunosorbent assays (ELISAs) were performed using a panel of DENV1-specific monoclonal antibodies (MAbs) against rDENV1ic and the parental wild-type strain see , binds i10.1128/mSphere.00380-16.1TABLE S1 TABLE S1, PDF file, 0.04 MB.Monoclonal antibody serotype specificity and epitopes. The serotype specificity, species, and epitopes of MAbs used in experiments are shown. Download Copyright © 2017 Gallichotte et al.2017Gallichotte et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the We also tested the binding of DENV serotype 2, 3, and 4 monoclonal antibodies  to rDENVWhile observing equivalent antibody binding between rDENV1ic and wild-type virus by ELISA, neutralization is an even more sensitive functional assay to compare viruses. Small changes in binding conformation may induce enhancement versus neutralization; as such, these assays must be equivalent between the infectious clone and the wild-type virus to justify the use of rDENVic for vaccine and immunogenicity studies. Using a focus reduction neutralization test FRNT), we examined the ability of well-defined MAbs to block virus infectivity. For all three DENV1 monoclonal antibodies , percent neutralization was nearly identical between the wild-type DENV1 and rDENV1ic  to C. SiRNT, we e10.1128/mSphere.00380-16.2FIG S1 FIG S1, PDF file, 0.2 MB.rDENV1ic is neutralized only by DENV1 serotype-specific antibodies in a focus reduction neutralization test. Neutralization of DENV1 and rDENV1ic by monoclonal antibodies was measured in U937+DC-SIGN flow cytometry neutralization assay using DENV1 serotype-specific MAbs 1F4, 14C10, and 1C19.2 (A to C) and DENV2, DENV3, and DENV4 serotype-specific antibodies 2D22, 5J7, and 5H2 (D to F). Download Copyright © 2017 Gallichotte et al.2017Gallichotte et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the To use the rDENVic to study antibody responses to specific epitopes, we generated a DENV1 mutant virus that incorporates the DENV4 5H2 epitope into the DENV1 backbone virus. 5H2 is a DENV4 type-specific and strongly neutralizing MAb isolated from a nonhuman primate infected with DENV4 , 25. A h50) but not complete ablation in binding of 1F4 to rDENV1ic-EDI . De. De50) bV1ic-EDI , and 14CV1ic-EDI . In contV1ic-EDI  27), ha, ha50) bV1ic-EDI . AdditioV1ic-EDI . In summ10.1128/mSphere.00380-16.3FIG S2 FIG S2, PDF file, 0.4 MB.14C10 epitope spans into EDIII from neighboring dimer. The 14C10 epitope sits primarily over EDI of a single envelope dimer, but it interacts with additional residues of EDIII in the adjacent envelope dimer. Download Copyright © 2017 Gallichotte et al.2017Gallichotte et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the in vitro  was constructed using a four-cDNA cloning strategy as previously described , 32, 38.For mutant virus generation, 12 predicted contact residues of 5H2 , 25 werel-glutamine and 2-mercaptoethanol. All media were additionally supplemented with 100 U/ml penicillin, 100 μg/ml streptomycin, and 0.25 μg/ml amphotericin B. All cells were incubated in 5% CO2.Cells were cultured as previously described by our group , 32. C6/Cell culture plates (24-well plates) were seeded with C6/36 cells to be confluent at the time of infection. The cell growth media were removed, and virus stocks were serially diluted 10-fold and then added to cells for 1 h at 32°C (C6/36) with gentle rocking. After incubation, cells were overlaid with 1% methylcellulose in Opti-MEM I (Gibco) supplemented with 2% FBS, nonessential amino acids, 100 U/ml penicillin, 100 μg/ml streptomycin, and 0.25 μg/ml amphotericin B and incubated at 32°C. After 4 to 6 days of incubation, the overlay was removed, and the cells were washed with phosphate-buffered saline (PBS) and fixed in 80% methanol. The cells were blocked in 5% nonfat dried milk in PBS (blocking buffer) and then incubated for 1 h at 37°C with anti-prM MAb 2H2 and anti-E MAbs 4G2 and DV1-E103 diluted in blocking buffer. The cells were washed two times with PBS and then incubated for 1 h at 37°C with horseradish peroxidase (HRP)-conjugated goat anti-mouse antibody (Sigma) diluted in blocking buffer. The plates were washed two times with PBS, and foci were developed using TrueBlue HRP substrate (KPL).C6/36 cells electroporated with rDENV1ic RNA were diluted in Opti-MEM I (Gibco), added to a confluent monolayer of C6/36 cells, overlaid with 1% methylcellulose, and incubated for 4 days. After incubation, cells were fixed and stained as described above. The percentage of electroporated cells capable of making viable infectious virus was calculated as follows: (number of foci/number of electroporated cells plated) × 100. To determine the amount of virus cells can produce, C6/36 cells were inoculated at a multiplicity of infection (MOI) of 0.01. Every 24 h, all cell culture supernatant was harvested (volume was replaced with fresh medium) and frozen at −80°C. The titers of the virus samples were determined as described above. To determine the kinetics at which cells become infected, C6/36 cells were inoculated at an MOI of 0.01. Every 12 h, cell culture medium was removed, and the cells were washed with PBS, fixed, permeabilized, and probed with anti-prM MAb 2H2 conjugated to Alexa Fluor 488. Infected cells were quantified using a Guava flow cytometer (Millipore). U937+DC-SIGN cells were infected at an initial infection of 2%, and every 12 h, a sample of cells were harvested, fixed, and stained as described for C6/36 cells.p-nitrophenyl phosphate substrate, and color changes were quantified by spectrophotometry as previously described  and blocked in 3% nonfat dried milk in TBST (blocking buffer), and equal quantities of virus (as previously titrated by enzyme-linked immunosorbent assay [ELISA]) were added and incubated for 1 h at 37°C. The plates were washed, and primary human MAbs were diluted fourfold in blocking buffer and added to the plates for 1 h. The plates were washed, and alkaline phosphate (AP)-conjugated secondary antibodies were added for 1 h at 37°C. The plates were washed and developed using escribed .4 U937+DC-SIGN cells for 2 h at 37°C. The cells were then pelleted, washed two times with fresh medium, and then incubated for 24 h. After incubation, the cells were fixed, permeabilized, and stained as described above for U937+DC-SIGN growth curves.For the focus reduction neutralization test (FRNT), 24-well cell culture plates were seeded with C6/36 cells to be confluent at time of infection. MAbs were diluted fourfold, mixed with ~45 focus-forming units (FFU) of virus, and incubated for 1 h at 32°C. After incubation, the virus-MAb mixture was added to C6/36 cells for 1 h at 32°C with gentle rocking. The overlay was added, and the cells were incubated for 4 to 6 days. The cells were fixed and stained as described above. For the flow-based neutralization assay, MAbs were diluted fourfold, mixed with virus , and incubated for 1 h at 37°C. After incubation, the virus-MAb mixture was added to 5 × 10U88535.1 and KJ160504.1. Monoclonal antibodies were synthesized from their deposited Protein Data Bank sequences (PDB identifiers [ID] shown in brackets) .Wild-type DENV1 and DENV4 sequences were derived from GenBank accession numbers


Samples were subject to chemical analysis for organic carbon (OC), elemental carbon (EC), and major water-soluble inorganic ions. The annual average PM2.5 mass concentration was 53 ± 36 μg·m−3 with the highest seasonal average concentration in spring and the lowest in summer. Water-soluble inorganic ions and carbonaceous aerosols accounted for 34% ± 15% and 33% ± 9%, respectively, of PM2.5 mass on annual average. The excellent, good, lightly polluted, moderately polluted, and heavily polluted days based on the Air Quality Index (AQI) of PM2.5 accounted for 40%, 42%, 11%, 4%, and 3%, respectively, of the year. The sum of the average concentration of sulfate, nitrate, and ammonium (SNA) increased from 4.2 ± 2.9 μg·m−3 during excellent days to 85.9 ± 22.4 μg·m−3 during heavily polluted days, and their contributions to PM2.5 increased from 15% ± 8% to 49% ± 10% accordingly. In contrast, the average concentration of carbonaceous aerosols increased from 9.2 ± 2.8 μg·m−3 to 51.2 ± 14.1 μg·m−3, and their contributions to PM2.5 decreased from 34% ± 6% to 29% ± 7%. Potential source contribution function (PSCF) analysis revealed that the major sources for high PM2.5 and its dominant chemical components were within the area mainly covering Shandong, Henan, and Hebei provinces. Regional pollutant transport from Shanxi province and Inner Mongolia autonomous region located in the west direction of SDZ was also important during the heating season.Daily PM The annual average PM2.5 exceeded the NAAQS in about 96% of the 74 cities. Among these cities, 13 are in the Beijing–Tianjin–Hebei (BTH) region, which had an annual average PM2.5 of 106 μg·m−3, much higher than that in Yangtze River Delta (YRD) region (25 cities with an average of 67 μg·m−3) and Pearl River Delta (PRD) region (9 cities with an average of 47 μg·m−3). The BTH region was evidently the most polluted region in China. PM2.5 has important effects on human health [2.5 chemical composition and sources in BTH is needed for assessing the effectiveness of the existing pollution control measures and for making future control measures to alleviate PM2.5 pollution.High levels of fine particles  located far northeast of the megacities  [42−, NO3−, NH4+, OC (organic carbon), and EC ) in PM2.5 at SDZ station were at similar levels to those at the urban sites in major cities such as Beijing, Tianjin, and Chengde [2.5 data at SDZ can to some extent provide regional characteristics of PM2.5 pollution in BTH. For this purpose, one-year continuous measurements of chemically-resolved PM2.5 data at SDZ were thoroughly analyzed to (1) systemically characterize PM2.5 levels and the dominant chemical components; (2) compare the chemical composition changes under different PM2.5 pollution levels; and (3) identify the possible source regions for dominant chemical components.To date, synchronic and continuous measurements of chemically-resolved PMazhuang) . Despite Chengde . Thus, aThe international Global Atmosphere Watch programme (GAW) was launched in 1989 to monitor atmosphere chemical compositions. At present, the GAW network includes more than 400 regional stations and 31 global stations which are mostly located in remote areas. SDZ station was the only regional station of GAW in BTH. SDZ station  is located in the north boundary of the North China Plain and is 100 km northeast of urban Beijing . The ins2.5 samples were collected using a particulate sampler  at a flow rate of 16.7 L·min−1. Samples were collected on 47 mm quartz fiber filter (Whatman QM-A). Sampling duration was 23 h starting at 9:00 local time each day and ending at 8:00 the following day. A total of 348 PM2.5 samples and 24 blank samples were collected from January to December 2015. The sampling for the whole year was divided into four seasons: spring (March–May), summer (June–August), autumn (September–November), and winter . Filter backgrounds were used to account for any artifacts caused by gas absorption, evaporation of semi-volatile compounds, and background filters. The net mass of samples ranged from 335 to 5552 μg, while the net mass of the blank filters accounted for about ±20 μg. The uncertainty of PM2.5 mass concentration ranged from ±0.4% to ±6%. Thus, the uncertainties caused by the Quartz filters are negligible. The aerosol-loaded filter samples were stored in a freezer at −18 °C before analysis to prevent the volatilization of particles. Quartz filters were also measured gravimetrically for calculating PM2.5 mass concentration.PM42−, NO3−, Cl−, Na+, NH4+, K+, Mg2+, and Ca2+) of each quartz filter were determined by ion chromatography  [OC and EC fractions of each quartz filter were analyzed using a DRI model 2001 carbon analyzer  following the IMPROVE_A thermal/optical reflectance (TOR) protocol . Eight wCA, USA) . Procedui × j small equal grid cells. The PSCF value in the ijth cell is estimated by mij/nij, which represents the probability of a “polluted” source from the ijth grid cell. Here, nij is defined as the number of endpoints that fall in the ijth grid cell and mij is defined as the number of “polluted” trajectory endpoints in the ijth grid cell. The resolution of the grid cell is 1° × 1°.To investigate the air mass origins of the air pollutants arriving at SDZ station, 72 h backward trajectories ) were calculated at an elevation of 500 m for every day of 2015 using the HYbrid Single-Particle Lagrangian Integrated Trajectory (HYSPLIT) 4 model  with meteorological input data from FNL . Potential source contribution function (PSCF) was also used to identify possible regional sources based on the HYSPLIT model (2:00 UTC). The zone of backward trajectories is divided into 2.5 mass concentration was 53 ± 36 μg·m−3 at SDZ station in 2015 (2.5 (35 μg·m−3). The meteorological data including precipitation and sunshine duration in 2015 were only slightly higher than those in 2009–2010. Although meteorological conditions in 2015 were in favor of the diffusion or scavenging of air pollutants, the annual average PM2.5 mass concentration in 2015 was 26% lower than the filter-based measurements (72 μg·m−3) during 2009–2010 [2.5 in BTH also evidently decreased from 98 μg·m−3 in 2013 to 76 μg·m−3 in 2015, suggesting a similar temporal trend between SDZ and BTH. Compared to other regional sites in China, the annual average PM2.5 at SDZ was slightly lower than at Lin’an  in YRD [−3, 750 m a.s.l.), in Hubei province [−3, <10 m a.s.l.), in PRD [2.5 between these regional sites were similar to those between the urban sites in these regions [2.5 pollution.The annual average PM in 2015 , which w009–2010 . Meanwhi) in YRD , slightl, in PRD . The spa regions , suggest2.5 seasonal patterns were found between 2009 and 2010  filter measurements and 2005–2007 online data at SDZ [2.5 concentration was higher in spring and summer than autumn and winter in previous studies, which was caused by frequent southern winds in spring and summer since the majority of air pollutant sources were located south of BTH [−3) and winter (57 ± 42 μg·m−3) and lower concentrations in autumn (50 ± 36 μg·m−3) and summer (42 ± 22 μg·m−3)  . The hig2.5 mass on annual average without evident seasonal variation (31%–36%) in 2015 at SDZ. SO42−, NO3−, and NH4+ (SNA) were the dominant water-soluble inorganic ions, accounting for 77% ± 16% of the total ions mass. The annual average SO42−, NO3−, and NH4+ were 38%, 48%, and 16%, respectively, lower in 2015 than in 2009–2010 [2.5 in the past five years at SDZ. Meanwhile, the annual average SO2, NO2, and O3 in 2015 in BTH were reduced by 47%, 15%, and 2%, respectively, compared with those in 2013. Generally, the formation of SO42− was mainly related with heterogeneous reactions of SO2. NO3− originated from heterogeneous reactions between HNO3 and NH3 during the daytime and hydrolysis ofN2O5 during the nighttime. The formation of HNO3 and N2O5 were related with the oxidation of NOx. The reductions of SO42− and NO3− should mainly be attributed to the reductions of their respective gaseous precursors.Water-soluble inorganic ions accounted for 34% ± 15% of PM009–2010 . The red42− and NH4+ concentrations were smaller than 30%, but more than a factor of 2.0 in NO3− concentrations. The highest and lowest seasonal concentrations of SO42− were observed in summer and winter, respectively, mainly due to the different air mass origins in different seasons as discussed below in 3− and NH4+ were the lowest in summer due to high ambient temperature, and differed little in the other seasons. As expected, opposite trends were seen from monthly variations between temperature and NO3− or NH4+  and between NH4+ and NO3− in all (R2 > 0.84) but the summer season (R2 = 0.76)  were found between NH4+ and the sum of SO42− and NO3− in all the seasons with regression slopes ranging from 0.84 to 0.88 (4+ mainly associated with both SO42− and NO3−. According to the thermodynamic equilibrium model (ISORROPIA II), NH4+ was insufficient to neutralize both SO42− and NO3−, and other cations  were also associated with these two anions  [To calculate the ionic balance of PMobserved , which i = 0.76) a,b. This to 0.88 c. These s, 2007) .2.5, accounting for 17% ± 5% and 3% ± 1%, respectively, of PM2.5 mass on annual average. The converting factor between organic matter (OM) and OC was chosen to be 1.8 due to the evident effect of biomass burning in BTH [2.5 mass. The annual average concentration of carbonaceous aerosols was 27% lower than that measured during 2009–2010 [2.5 reduction during the same period (26%).OC and EC were important fractions of PM009–2010 , similar2.5 was similar to that of carbonaceous aerosols, higher than that of NH4+, but much lower than those of SO42− and NO3−. Thus, only reducing SO2 and NOx emissions may not be very effective in reducing PM2.5 in areas with NH3 limited conditions, such as the case presented here. More attention should be paid to NH3 emission control in the future.From the above discussions it can be seen that the percentage reduction of PM−3 in spring to 1.9 μgC·m−3 in winter. The correlation between OC and EC has been frequently used to identify possible sources of carbonaceous aerosols  [R2 ranging from 0.50 to 0.65) in spring, summer, and winter and a good correlation (R2 = 0.84) in autumn were found between OC and EC  . ModeratC and EC a. The sln summer b. The in for BTH  showed m for BTH a, the in2.5 from the determined chemical components, including water-soluble inorganic ions and carbonaceous aerosols, accounted for 67% ± 15% of measured PM2.5 mass. The reconstruction efficiency ranged from 63% ± 15% in spring to 70% ± 17% in winter. The lowest reconstruction efficiency in spring was likely due to the frequent fugitive dust events with high Ca2+ concentrations  (HJ633-2012), daily PM5 at SDZ a. On a s2.5 increased from 4.2 ± 2.9 μg·m−3 to 85.9 ± 22.4 μg·m−3 and from 9.2 ± 2.8 μg·m−3 to 51.2 ± 14.1 μg·m−3, respectively, when PM2.5 AQI increased from excellent to heavy polluted cases  were 4.7, 5.3, 6.1, and 5.7 times of those under clean conditions (including excellent and good days) in spring, summer, autumn, and winter, respectively; while 2.0, 1.9, 2.8, and 3.1 times, respectively, for carbonaceous aerosols. Evidently, SNA increased much faster than carbonaceous aerosols when PM2.5 AQI increased from excellent to heavy polluted cases.On a seasonal scale, the concentrations of SNA in PM2.5 mass evidently increased from 23% ± 12%, 28% ± 17%, 24% ± 15%, and 20% ± 11% during the clean days to 42% ± 12%, 63% ± 9%, 53% ± 6%, and 39% ± 11% during the polluted days in spring, summer autumn, and winter, respectively; while for carbonaceous aerosols, decreased from 32% ± 9%, 30% ± 6%, 36% ± 6%, and 39% ± 10% to 24% ± 5%, 19% ± 3%, 30% ± 5%, and 38% ± 8%, respectively. The larger increases in the percentage contributions of SNA in summer and autumn than in spring and winter were likely caused by higher relative humidity (RH) in summer and autumn  sources were located [2.5 were observed in summer, the highest concentrations of SO42− were also observed in summer. In fact, the highest concentrations of SO42− were also observed in summer in several megacities  located in the south region of BTH [42− gradually decreased from southern cities (Tianjin and Shijiazhuang) to a northern city (Beijing) within the region. As discussed in 3− were observed in summer due to the highest ambient temperatures. Moreover, the lowest concentrations of carbonaceous aerosols were also observed in summer, suggesting residential heating and biomass burning instead of industrial sources south of BTH were the crucial sources of carbonaceous aerosols.Air mass paths in summer originated from four directions including south, northeast, north, and west. The frequency of air masses from south of BTH was 36% in summer, where many air pollutants . The highest seasonal OC and EC concentrations in winter were probably caused by regional heating sources, as supported by the fact that the highest monthly average OC and EC were observed in the heating season from the middle of November to the middle of March next year . Although the locations of the higher values or “polluted spots” were different for PM2.5 and the dominant chemical components, the “polluted spots” mainly originated from the south to west directions  . Other “2.5 pollution seemed to have alleviated in BTH in recent years, as seen from the 26% decrease in the annual average PM2.5 and 34% decrease in the sum of SNA and carbonaceous aerosols at SDZ station in 2015 compared to those during 2009–2010. However, the annual PM2.5 mass concentration at SDZ was still 51% higher than the NAAQS in 2015, which was largely caused by high concentrations of NO3−, SO42−, and NH4+ during polluted days, especially the increase of NO3− during heavily polluted days. Regional transport of air pollutants originating from the south to west regions of SDZ was the most likely source of PM2.5, especially SO42−. Open biomass burning activities and heating  in BTH likely contributed to the high concentrations of carbonaceous aerosols at SDZ station. While there is a need to further decrease NOx and SO2 emissions in order to reduce the number of heavily polluted days in this region, decreasing NH3 emissions might be more effective since the area is under a NH3-limited scenario in the formation of PM2.5. Additional modeling sensitivity studies are needed to investigate the non-linearity between gaseous-precursors emissions reduction and PM2.5 formation in this region for providing scientific guidance on making future emission control policies.PM


Acute lung injury and acute respiratory distress syndrome (ALI/ARDS) is a severe clinical syndrome with mortality rate as high as 30–40%. There is no treatment yet to improve pulmonary endothelial barrier function in patients with severe pulmonary edema. Developing therapies to protect endothelial barrier integrity and stabilizing gas exchange is getting more and more attention. Sphingosine-1-phosphate (S1P) is able to enhance the resistance of endothelial cell barrier. S1P at physiological concentrations plays an important role in maintaining endothelial barrier function. Proliferation, regeneration and anti-inflammatory activity that mesenchymal stem cells (MSCs) exhibit make it possible to regulate the homeostatic control of S1P.By building a pulmonary endothelial cell model of acute injury, we investigated the regulation of S1P receptors and sphingosine kinases expression by MSCs during the treatment of acute lung injury using RT-PCR, and investigated the HPAECs Micro-electronics impedance using Real Time Cellular Analysis.α expression was more significant when MSC was used in combination with S1P. The combination effection mainly worked on S1PR2, S1PR3 and SphK2. The results show that when MSCs were used in combination with S1P, the selectivity of S1P receptors was increased and the homeostatic control of S1P concentration was improved through regulation of expression of S1P metabolic enzymes.It was found that the down-regulation of TNF-The study found that, as a potential treatment, MSCs could work on multiple S1P related genes simultaneously. When it was used in combination with S1P, the expression regulation result of related genes was not simply the superposition of each other, but more significant outcome was obtained. This study establishes the experimental basis for further exploring the efficacy of improving endothelial barrier function in acute lung injury, using MSCs in combination with S1P and their possible synergistic mechanism. Acute lung injury and acute respiratory distress syndrome (ALI/ARDS) was first recognized as a clinical syndrome in the 1960s. It manifests as severe and acute respiratory failure with hypoxemia and lung infiltration and is often caused by pneumonia, sepsis and major trauma. The mortality rate of ALI/ARDS is as high as 30–40% . In the in vivo. It is mainly present in plasma and tissues and is able to enhance the resistance of endothelial barrier. Endothelial barrier enhancement mediated by S1P is completed by activating the Gi and Racl signaling pathway through S1P receptor (Sphingosine-1-phosphate (S1P) is a ubiquitous sphingomyelin and is an important regulator of vascular endothelial cell permeability and fluid balance receptor . Studiesreceptor . FTY720,receptor . This inSeveral studies have demonstrated that S1P, at physiological concentration, has an important role in maintaining endothelial barrier function. For S1P and its structure analogues, their limitations in therapeutic function, confirm the conception of sphingolipid homeostasis. The fate of the cells is determined by the homeostasis of S1P concentration . As an eRecent clinical studies have found that it is unlikely for any single drug to reverse the severe pathological injury caused by acute lung injury. Therefore, in the therapy of ALI/ARDS, there has been attention focused on cell therapy, especially mesenchymal stem cell therapy.Mesenchymal stem cells (MSCs) are multi-functional cells with the ability of self-renewal. They were first discovered in bone marrow and can differentiate into bone, cartilage, fat, muscle and other tissues. At present, MSCs have demonstrated great efficacy in the treatment of cirrhosis , systemiin vivo is a prerequisite to improving endothelial barrier and treating acute lung injury. The anabolic process of S1P is regulated by many cytokines. The outcomes of many diseases when MSCs were used demonstrate that it can secrete various growth and inflammatory factors, playing a role in immune regulation. Besides, according to some studies, S1P is capable of promoting the differentiation of MSCs in vitro  purchased from Sigma-Aldrich Company was dissolved in methanol and kept at −20 °C. Lipopolysaccharide (LPS) E.coli O55:B5 purchased from Sigma-Aldrich Company was dissolved in saline and kept at −20 °C.β-actin was purchased from Sigma-Aldrich.Anti-EDG-1 (S1PR1), Anti-EDG-5 (S1PR2) and Anti-EDG-3 (S1PR3) were purchased from Santa Cruz Biotechnology. Anti-5U/L penicillin and 100 mg/L streptomycin. Collagenase IV solution was added to digest the cells, at 37 °C for 2 h. Single cell suspension of MSCs was obtained following filtration. After the cells were counted, the suspension was inoculated into 10 ml MSC medium  in a 100 mm petri dish with a cell density of 2.5 to 4 × 104/cm2. After primary culture at 37 °C and 5% CO2 in an incubator for 7 to 9 days, first passage was carried out according to overall growth condition and local density. Incubation was continued until the density exceeds 80%, repeated the above operations. Subculture generations at P3 to P5 were used for subsequent experiments. Human pulmonary artery endothelial cells (HPAECs) purchased from ScienCell Company were cultured in endothelial cell medium  at 37 °C in a 5% CO2 in incubator. P5 to P8 cells were used for subsequent experiments.Newborn umbilical cord was taken under sterile conditions  and remnant blood in umbilical vein and artery was rinsed in D-HANKS solution containing 104 per well in 100 µl, then cultured in an incubator at 37 °C and 5% CO2 for 12 h following which LPS was added at varying concentrations of 0, 0.5, 1, 2 and 5 µM. The 16-well E-Plates were placed within the Real Time Cellular Analysis (RTCA) System (ACEA Biosciences) and cultured in an incubator at 37 °C and 5% CO2. Micro-electrical impedance of HPAEC cells were detected in real-time to investigate the effects of acute injury caused by LPS on HPAEC cells, at different concentrations.HPAEC cells between P5 to P8 were inoculated into a 16-well E-Plate  at a concentration of 2 × 105 per well in 600 µl, then cultured in incubator at 37 °C and 5% CO2 for 6 to 12 h after LPS was added at a final concentration of 1 µM. Cells were collected at different time points and the expression of TNF-α was detected to investigate the acute injury effects of LPS on HPAEC cells.HPAEC cells between P5 to P8 were inoculated into a 24-well cell culture plate  at a concentration of 1 × 104 per well and then LPS at a concentration of 1 µM was added. In the meantime, 60 µl of MSCs at different proportions  were inoculated into a 16-well E-Plate Insert (ACEA Biosciences), The insert was then placed into a receiver plate containing 100 µl of MSC medium. The 16-well E-Plates were placed within the RTCA System, together with the receiver plates, and were cultured in an incubator at 37 °C and 5% CO2 for 12 h. Following that, medium in the E-Plates was discarded and replaced with 100 µl of fresh endothelial cell medium, and the insert was filled to 60 µl with MSC medium in the corresponding receiver plates. The inserts were then placed into the E-Plates containing HPAECs and co-cultured within the RTCA System at 37 °C and 5% CO2 for 8–24 h. Micro-electrical impedance of HPAEC cells were detected in real-time to investigate the effect of MSCs on HPAEC cell injury.HPAEC cells between P5 to P8 were inoculated into a 16-well E-Plate (ACEA Biosciences) in 100 µl at a concentration of 2 × 105 per well following which LPS at a concentration of 1 µM was added. Meanwhile, 100 µl of MSCs with different proportions  were inoculated into the upper compartment of a 24 well Transwell plate , and 600 µl of MSC medium was added into the lower compartment. The 24-well cell culture plates and Transwell plates were incubated at 37 °C and 5% CO2 for 12 h. Then the medium in the cell culture plates was discarded and replaced with 600 µl of fresh endothelial cell medium, the upper compartment of the Transwell plates was filled to 100 µl with MSC medium in the corresponding lower compartments. After 8 h to 24 h of co-culture with the upper compartment of Transwell plates placed into cell culture plates, HPAEC cells were collected. The effect of MSCs on HPAEC cell injury was investigated by determining the expression change of TNF-α.HPAEC cells between P5 to P8 in 600 µl medium were inoculated into a 24-well cell culture plate at a concentration of 1 × 102. Detection was determined according to the manufacturer’s instructions.A 16-well E-Plate was placed on Xcelligence RTCA DP system (ACEA Biosciences) and incubated at 37 °C and 5% CO® Mini Kit  according to manufacturer’s instructions. With 1 µg of RNA as template, reverse transcription reaction was performed using QuantScript RT Kit  according to manufacturer’s instructions. Utilizing Bio-Rad iQ™5 Multicolor Real-Time PCR Detection System and with GAPDH as internal reference, fluorescence intensity was detected using SuperReal PreMix SYBR Green  according to the manufacturer’s instructions. The primers used are shown in Total RNA was extracted from collected HPAEC cells using RNeasyg, room temperature for 10 min and the supernatant was collected. After protein concentration was measured using BCA method, 20 to 50 µg of protein was separated by 12% SDS-PAGE, then transferred to a PVDF membrane and sealed with TBST buffer containing 5% BSA. Specific antibodies were added and incubated overnight. On the next day, after washing and secondary antibody incubation, chromogenic assay was performed using ECL chemiluminescence kit .The collected HPAEC cells were washed with PBS twice. Then 200 µl lysis buffer was added, which contains 20 mM Tris–HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM pyrophosphate sodium phosphate, 1 µg/ml leupeptin, 1 µg/ml aprotinin and protease inhibitors . Cell lysate was centrifuged at 10,000 × p < 0.05 indicates that the difference is statistically significant.All experimental data are represented as mean ± standard deviation (mean ± SD). One-way ANOVA (Dunnett test) was adopted in multiple group comparison. Data was analyzed with software GraphPad Prism 6.0. α is significantly increased , groups with inoculation ratio of HPAECs and MSCs 1:1, 1:2 and 1:4. They were non-contact co-cultured with HPAECs following treatment with LPS. Cells of each group were collected and the change in expression of TNF was 1:2 . Results was 1:2 . The resα was determined. Results show that compared to MSCs alone, the down-regulation of TNF-α expression was more significant when S1P was combined with MSCs (Sphingosine-1-phosphate (S1P) is a biologically active ubiquitous sphingomyelin. It is mainly produced by platelets and also present in plasma and tissues. When its physiological concentration is in the range of 0.2 to 1.1 µM, it can effectively enhance the endothelial barrier function. In this study, a HPAEC acute injury model was established using 1 µM LPS and treated with S1P at final concentrations of 0 µM, 0.5 µM, 1 µM, 2 µM, and 5 µM. The effect of LPS on HPAEC injury was studied by RTCA experiment. Results show that, S1P within physiological concentrations can effectively enhance the micro-electrical impedance of HPAECs . In addiith MSCs .In order to further investigate the possible synergistic mechanism of MSC and S1P in the treatment of HPAEC injury, the change in S1P receptors and sphingosine kinase expression in HPAECs treated with MSCs alone or the combination of MSCs with S1P, were determined using RT-PCR. After 12 h following exposure to LPS, the expression changes of S1P receptors  and sphingosine kinases (SphK1 and SphK2) in HPAECs were studied using RT-PCR. Results demonstrate that the expression of target genes were dramatically increased following LPS induced injury. The expression of S1PR1 and S1PR3 were increased significantly, while that of S1P2 was not. There was no significant difference in the expression levels of SphK1 and SphK2 following LPS induced injury . These rα expression was significant when MSCs were used in combination with S1P. This indicated that there is a possible synergistic mechanism, between MSCs and S1P, in the treatment of acute injury . After 8 h of non-contact co-culture, HPAECs cells were collected and total RNA were extracted. The changes in expression of S1P related genes were detected using RT-PCR. Results show that compared with MSC alone, the down-regulation of TNF-e injury . The rege injury .In vivo, sphingomyelin (SM) is catalyzed to produce ceramide, which is further hydrolyzed to sphingosine. Sphingosine is able to produce S1P by phosphorylation catalyzed by sphingosine kinase (SphK1 and SphK2). Our study results demonstrate that the expression of sphingosine kinase (SphK1 and SphK2) differs in HPAECs following LPS injury. When MSCs were used alone, it mainly worked on SphK1, the expression level of which was significantly reduced. When MSCs were used in combination with S1P, the expression of SphK2 was also significantly reduced . After 8 h of non-contact co-culture, HPAECs cells were collected and total proteins were extracted. The changes in expression of S1P receptors were detected through Western-Blot. The results were consistent with that from the RT-PCR. The results of regulation of different S1P receptors were different when MSCs were added, where S1PR2 and S1PR3 were the main targets. The effect on S1PR1 was not significant whether MSCs were used alone or in combination with S1P .in vivo and is able to enhance endothelial barrier. Its homeostasis in vivo is tightly regulated by the balance between synthesis and degradation. In vivo, sphingomyelin (SM) is catalyzed by Sphingomyelinase to produce ceramide (Cer), which produces sphingosine in hydrolysis catalyzed by Neuraminidase. Phosphorylation of sphingosine produces S1P catalyzed by sphingosine kinase (SphK1 and SphK2). Physiological concentrations of S1P plays an important role in maintaining endothelial barrier function. It has been demonstrated that the activity of SphK1 and the dynamic concentration balance of SphK1/S1P axis, are necessary for the modulation of inflammatory signals and are related to the regulation of innate, adaptive and other immune cells. Studies have also shown that abnormal changes in SphK1 or S1P lead to many inflammatory and autoimmune diseases, including asthma, rheumatoid arthritis, sepsis, inflammatory bowel disease and so on (Sphingosine-1-phosphate (S1P) is an agonist mainly present in plasma and tissues. It is an important regulator of vascular endothelial cell permeability and fluid balance nd so on . Our stuStudies on S1P show that endothelial barrier enhancement mediated by S1P is completed by activating the Gi and Racl signaling pathway through the S1P receptor . There aClinical studies have shown that any single target and drug are not likely to reverse the severe pathological injury caused by ALI/ARDS and achieve great therapeutic effect quickly. Innovative multi-target synergistic treatment mechanism may be an effective method to solve this problem. In essence, stem cell therapy does not work on a single target or as a single drug in therapy, but on multiple targets based on biological distress signals from the diseased tissues and as multiple therapies. Our study found that when MSCs worked on HPEACs with acute injury, the expressions of S1P receptors and sphingosine kinase were regulated. This indicates that MSCs are able to affect the expression of multiple S1P related genes, improve endothelial barrier and cure acute lung injury. Meanwhile, as an endogenous bioactive molecule, S1P is an important regulator of vascular endothelial cell permeability and fluid balance. In the treatment of acute lung injury, it plays an important role in improving endothelial barrier by regulating the expression of key enzymes in anabolic processes and specific receptors on signaling pathways. Our study results demonstrate that both MSCs and S1P can alleviate acute lung injury. But the combined use of MSCs and S1P shows significant efficacy in the regulation of S1P related gene expression, suggesting that some synergism between MSCs and S1P may exist in the treatment of acute injury. Further studies are need to determine whether MSCs when used in combination with S1P, further improve the endothelial barrier and if a possible synergistic mechanism exists and maybe to provide a novel therapeutic strategy in the treatment of acute lung injury.In this study we built a pulmonary endothelial cell model of acute injury by LPS, and investigated the regulation of S1P receptors and sphingosine kinases expression by MSCs combined with S1P. The study results demonstrated that both MSCs and S1P can alleviate acute lung injury. When MSCs were used alone, only the expression of SphK1 was reduced, and there was no significant impact on the expression of SphK2. When MSCs were used in combination with S1P, the expression of SphK1 and SphK2 were reduced simultaneously. We also found that when MSCs works on acute injured HPAECs, the regulation of individual S1P receptors were different, which was mainly acted on S1PR2 and S1PR3. The expression of S1PR2 and S1PR3 were further reduced when MSCs were used in combination with S1P. These results show that MSCs play a role in the treatment of acute lung injury by regulating the expression and activity of S1P related genes, when MSC combined with S1P there is a possible synergistic mechanism exists.10.7717/peerj.2712/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.2712/supp-2Supplemental Information 2Click here for additional data file.10.7717/peerj.2712/supp-3Supplemental Information 3Click here for additional data file.10.7717/peerj.2712/supp-4Supplemental Information 4Click here for additional data file.10.7717/peerj.2712/supp-5Supplemental Information 5Click here for additional data file.10.7717/peerj.2712/supp-6Supplemental Information 6Click here for additional data file.