Regulation of cell growth and expression of 7B2, PC2, and PC1/3 by TGFbeta 1 and sodium butyrate in a human pituitary cell line (HP75)

Recent studies have shown that 7B2 and the neuroendocrine-specific proconvertase PC2 have important roles in pituitary cell proliferation and hormone secretion. Studies from our laboratory have also shown that TGFb1 regulates anterior pituitary cell proliferation and hormone secretion. To study the regulation of 7B2 in human pituitary tumors, we used a cell line derived from a human pituitary adenoma (HP75) that has been shown to express 7B2, PC1, PC2, and TGFβ receptors to analyze the effects of TGFβ1 and the histone deacetylase inhibitor (HDACI) sodium butyrate (NaB) treatment on 7B2 mRNA expression along with the neuroendocrine-specific proconvertases 1/3 (PC1) and PC2 mRNA and protein expression. RNA was quantified by real-time PCR and proteins were detected by immunohistochemistry and Western blotting. Treatment of cells with 1 mM NaB or 1 nM TGFβ1 for 4 d decreased cell proliferation with a concomitant increase in the cell cycle protein p21. Real-time PCR analysis showed a significant increase in 7B2 mRNA after NaB and TGFβ1 treatment. PC2 mRNA was down regulated by NaB while PC1 mRNA was unchanged. TGFβ1 stimulated PC1, but not PC2 mRNA levels. Changes in PC1 and PC2 protein were similar to changes in the mRNAs, but the differences were not significant. These results indicated that NaB and TGFβ1 inhibit pituitary cell proliferation and regulate the expression of 7B2, PC1, and PC2 in a cell culture model of pituitary tumors. Our results also indicate that inhibition of pituitary cell proliferation is associated with increased expression of 7B2 mRNA.     

Tetra-fluorinated aromatic azide for highly efficient bioconjugation in living cells

We developed a fast strain-promoted azide–alkyne cycloaddition reaction (SPAAC) by tetra-fluorinated aromatic azide with a kinetic constant of 3.60 M⁻¹ s⁻¹, which is among the fastest SPAAC ligations reported so far. We successfully employed the reaction for covalent labelling of proteins with high efficiency and for bioimaging of mitochondria in living cells. The reaction could be a generally useful toolbox for chemical biology and biomaterials.

A fast strain-promoted azide–alkyne cycloaddition based on tetra-fluorinated aromatic azide was developed and applied to label proteins and living cells with high efficiency.

Strain-promoted azide–alkyne cycloaddition (SPAAC) as an efficient bioconjugation tool has recently found a wide range of applications in chemical biology, materials science and medical science.^1–3 In 2001, Sharpless et al., first introduced the concept of click chemistry that emphasized on the rapid synthesis of useful new compounds and combinatorial libraries through heteroatom links (C–X–C).⁴ To overcome the limitations for classical copper-catalyzed azide–alkyne cycloaddition (CuAAC),⁵ Bertozzi improved the method using strained cyclooctynes to react with azide in 2004, which effectively avoids the transition metal catalysts (such as Cu) and makes it more general for bioconjugations.^6,7 It gave birth to the development of SPAAC. But one of the limitations is the relative low reaction rate compared with other biorthogonal reactions and a high rate could be beneficial for labelling low abundance or transient structures with short-lived signal molecules, such as radioisotopes for imaging.⁸ During the past decade, efforts have been made to further improve the method. Structure modified cyclooctynes, including aliphatic cyclooctynes⁹ and (di)benzoannulated cyclooctynes¹⁰ have been investigated to accelerate the reaction, and recently the modified azides was also reported to remarkably increase SPAAC rate.¹¹We have been interested in the development of fast chemoselective reactions for bioconjugations.¹² We reported o,o′-difluorinated aromatic azide can accelerate both the reaction rate significantly on SPAAC and H₂S-mediated reduction of the azide.^12c Therefore, we envision that multi-fluorinated aromatic azide may have interesting properties for fast and convenient bifunctional conjugation. 4-Azido-2,3,5,6-tetrafluorobenzoic acid (1) was selected for investigation. Here, we report the SPAAC kinetic properties of 1 and its applications for protein labelling in vitro and for bioimaging of subcellular organelles in living cells (Fig. 1).Open in a separate windowFig. 1Rational design of SPAAC reaction based on multi-fluorinated aromatic azide. (a) SPAAC reaction based on o,o′-difluorinated aromatic azide reported in our previous work; (b) SPAAC reaction based on tetra-fluorinated aromatic azide for faster and bifunctional ligation.To test our idea, the reaction between 1 and [1R,8R,9S]-bicyclo[6.1.0]non-4-yn-9-yl methanol (2) was studied for SPAAC reaction, monitored with time-dependent ¹H NMR. To our delight, the reaction could finish within 5 min. As shown in Fig. 2, the ¹H-NMR signal of 2 (40 mM) completely changed to the product (3) after adding 1 for 5 min, and there''s no further change at 15 min and 2 hours in NMR spectra. The resulted product was also confirmed by high resolution mass spectrum. This result implies that the 4-azido-2,3,5,6-tetrafluorobenzoic acid derivatives can be used for this fast SPAAC reaction. But NMR is limited by its relative low sensitivity for accurate testing the kinetic rate at micromolar concentration.Open in a separate windowFig. 2 ¹H NMR analysis of the reaction between 1 (120 mM) and 2 (40 mM). The reaction was carried out in CD₃OD.In order to quantify the kinetic rate, we designed a procedure based on fluorescence resonance energy transfer (FRET) method to monitor this fast SPAAC reaction. We synthesized the azide compound 4 conjugated to an azo-quencher and the cyclooctyne compound 5 conjugated to a BODIPY-dye (Fig. 3). All the compounds were isolated and characterized by NMR and HRMS. After the reaction between 4 and 5, 6 could formed in which BODIPY fluorescent signal was quenched due to the FRET effect. Such fluorescence change could be employed to monitor the reaction in a real-time.Open in a separate windowFig. 3(a) Chemical structures of 4 and 5 and their SPAAC reaction to produce 6. (b) Time-dependent fluorescence spectra of 5 (2 μM) upon treated with 4 (30 μM) in PBS (50 mM, pH 7.4, containing 70% CH₃CN) at room temperature (excitation, 473 nm). The reaction time is shown inset. (c) The linear relationship between the concentration of 4 and k_obs. The slope of the best linear fitting gives the reaction rate k₂ (M⁻¹ s⁻¹).The reaction between 4 and 5 was set up in PBS buffer (2 μM 5, 50 mM, pH 7.4) and the maximum emission at 511 nm was monitored with the excitation at 473 nm. The pseudo-first-order rate k_obs was determined by fitting the data with a single exponential function. The linear fitting between k_obs and the concentrations of 4 gave the reaction rate (k₂) as 3.60 M⁻¹ s⁻¹. It''s two-fold faster than o,o′-difluorinated aromatic azide that we reported earlier, 1500-fold faster than the original SPAAC reaction and among the fastest SPAAC reactions reported so far.With this highly efficient reaction, covalent protein labelling was first tested as its application. N-Hydroxysuccinimide ester of 4-azido-2,3,5,6-tetrafluorobenzoic acid was synthesized as bifunctional labelling compound (7) for amide and SPAAC reactions. We chose bovine serum albumin (BSA) and lysozyme as model proteins considering their different sizes and functions.⁸ As shown in Fig. 4a, we tested to label protein first with 7 and then conjugated a dye to the protein using fluorescent cyclooctyne (5). BSA or lysozyme was treated with 0.5 mM 7 in PBS buffer (50 mM, pH 8.5, containing 10% DMSO) for 2 h to incorporate tetra-fluorinated aromatic azide into the protein. After removing of small molecules, the azide labelled protein was incubated with 1 mM 5 for another 2 h to achieve SPAAC protein fluorescent dye labelling. As a control, the azide labelled protein was also treated with Na₂S for 10 minutes to reduce azide into amine before incubating with 5, so to prove the reaction specificity. The labelled proteins were analysed with SDS-PAGE either stained by Coomassie blue or excited under UV lamp to visualize the desired protein. The results are shown in Fig. 4b. The strong fluorescent labelled BSA (lane 1) and lysozyme (lane 4) could be observed after the reaction with tandem addition of 7 and 5, while the controls of 5 only (lane 2 and 5) and Na₂S treated labelling (lane 3 and 6) did not show any fluorescent signal. The results indicated that tetra-fluorinated aromatic azide-cyclooctyne is a highly efficient SPAAC reaction for protein labelling.Open in a separate windowFig. 4Fluorescence labeling of BSA and lysozyme via the SPAAC reaction. (a) The fluorescence labelling strategy. (b) 15% SDS-PAGE of BSA (lane 1–3) and lysozyme (lane 4–6) was imaged under UV lamp (right) and then stained by Coomassie blue (left).To future explore the biocompatibility of this SPAAC reaction in living cells, we tested it for bioimaging of the subcellular organelle mitochondria. Triphenylphosphines are known to enrich into mitochondria mainly due to its positive charged character.¹³ We designed and synthesized compound 8, with the azide conjugated to a triphenylphosphine. Upon incubating 8 with the cells, we expected it would target mitochondria, so we can use SPAAC reaction to label the cells with fluorescent compound 5 (Fig. 5a). Human embryonic kidney cells 293 (HEK 293) were chosen for the experiment. The cells were first treated with 8 (10 μM) for 20 min to accumulate tetra-fluorinated aromatic azide into mitochondria, and then incubated with 5 μM 5 for another 30 min. After the non-specific fluorescent signal was washed away, the cells were imaged under the excitation of 488 nm. The experimental conditions and results were shown in Fig. 5b. The cells could only be fluorescence-labelled when treated with both 8 and 5, and the imaging signal all localized on mitochondria. The result proved both the feasibility and biocompatibility for the application of this improved SPAAC conjugation at cellular level. Its superior kinetic character (k₂ of 3.60 M⁻¹ s⁻¹) may even enable in vivo applications for pretargeted imaging,¹⁴ which is currently under investigation.Open in a separate windowFig. 5Fluorescence labelling of mitochondria in living cells via the SPAAC reaction. (a) The fluorescence labelling strategy. (b) Fluorescence images of HEK293 cells. HEK293 cells were treated for 30 min only with 10 μM of 5 (left); 20 min only with 10 μM of 8 (middle); 20 min with 10 μM of 8 followed by 5 μM of 5 for 30 min (right).     

A novel 3D Cd(ii) coordination polymer generated via in situ ligand synthesis involving C–O ester bond formation

A novel 3D Cd(ii) coordination polymer {[Cd(ddpa)(2,2′-bpy)]·H₂O}_n (1) (H₂ddpa = 5,10-dioxo-5,10-dihydro-4,9-dioxapyrene-2,7-dicarboxylic acid, 2,2′-bpy = 2,2′-bipyridine) is hydrothermally synthesized in situ, and the influencing factors and mechanism for the in situ reaction are briefly discussed. The synthesis of 1 requires the formation of a new C–O ester bond. This current study confirms that metal ions and N-donor ligands play important roles in the domination of the in situ ligand from 6,6′-dinitro-2,2′,4,4′-biphenyltetracarboxylic acid (H₄dbta). Furthermore, the structure, thermal stability and photoluminescent property of 1 are also investigated.

A 3D Cd(ii) coordination polymer comprising ligand molecules not included in the original reaction mixtures but instead formed via in situ ligand synthesis involving a C–O ester bond.     

Low temperature CO oxidation catalysed by flower-like Ni–Co–O: how physicochemical properties influence catalytic performance

In this work, mesoporous Ni–Co composite oxides were synthesized by a facile liquid-precipitation method without the addition of surfactant, and their ability to catalyse a low temperature CO oxidation reaction was investigated. To explore the effect of the synergetic interaction between Ni and Co on the physicochemical properties and catalytic performance of these catalysts, the as-prepared samples were characterized using XRF, XRD, LRS, N₂-physisorption (BET), SEM, TEM, XPS, H₂-TPR, O₂-TPD and in situ DRIFTS characterization techniques. The results are as follows: (1) the doping of cobalt can reduces the size of NiO, thus massive amorphous NiO have formed and highly dispersed on the catalyst surface, resulting in the formation of abundant surface Ni²⁺ ions; (2) Ni²⁺ ions partially substitute Co³⁺ ions to form a Ni–Co spinel solid solution, generating an abundance of surface oxygen vacancies, which are vital for CO oxidation; (3) the Ni_0.8Co_0.2 catalyst exhibits the highest catalytic activity and a satisfactory stability for CO oxidation, whereas a larger cobalt content results in a decrease in activity, suggesting that the amorphous NiO phase is the dominant active phase instead of Co₃O₄ for CO oxidation; (4) the introduction of Co can alter the morphology of catalyst from plate-like to flower-like and then to dense granules. This morphological variation is related to the textural properties and catalytic performance of the catalysts. Lastly, a possible mechanism for CO oxidation reaction is tentatively proposed.

The flower-like catalyst possesses highly dispersed amorphous NiO and a high concentration of surface oxygen vacancies which are the central points for CO oxidation.     

Promising bactericidal approach of dihydrazone analogues against bio-film forming Gram-negative bacteria and molecular mechanistic studies

Gram-negative members of the ESCAPE family are more difficult to treat, due to the presence of an additional barrier in the form of a lipopolysaccharide layer and the efficiency of efflux pumps to pump out the drugs from the cytoplasm. The development of alternative therapeutic strategies to tackle ESCAPE Gram-negative members is of extreme necessity to provide a solution to the cause of life-threatening infections. The present investigations demonstrated that compounds 17, 20, 25 and 26 possessing the presence of electron donating (OH and OCH₃) groups on the phenyl rings are highly potent; whereas compounds 9, 10, 15, 16, 18, 33 and 36 showed moderate activity against Gram-negative bacteria. An excellent dose-dependent antibacterial activity was established compared to that of the standard antibiotic ampicillin. Significant anti-biofilm properties were measured quantitatively, showing optical density (O.D) values of 0.51 ± 015, 0.63 ± 0.20, 0.38 ± 0.07 and 0.62 ± 0.11 at 492 nm and the leakage of cellular components by the compounds, such as 17, 20, 25 and 26, increased the O.D. of respective treated samples compared to the control. In addition, the implication of experimental results is discussed in the light of the lack of survivability of planktonic bacteria and biofilm destruction in vitro. These results revealed the great significance of the development of a new generation of synthetic materials with greater efficacy in anti-biofilm properties by targeting to lock the bio-film associated protein Bap in Gram-negative bacteria.

Gram-negative members of the ESCAPE family are more difficult to treat, due to the presence of an additional barrier in the form of a lipopolysaccharide layer and the efficiency of efflux pumps to pump out the drugs from the cytoplasm.     

Improved performance of optical phased arrays assisted by transparent graphene nanoheaters and air trenches

In this paper, high-performance optical phased arrays (OPAs) assisted by transparent graphene nanoheaters and air trenches have been designed and simulated. By directly locating graphene nanoheaters on silicon waveguides, heating efficiency is enhanced by 62.96% compared to conventional structures with 1 μm SiO₂ overlays, and is further enhanced by a factor of 200% by the presence of air trenches. Thanks to the high thermal conductivity of graphene, a record-high operation speed on the order of 200 kHz is realized. Power consumption for π phase shift is 4.65 mW, approximately half of that of the state-of-the-art OPAs. By introducing air trenches, thermal crosstalk is significantly reduced, resulting in an enlarged fill factor. In addition, a novel beam steering scheme in the θ direction is proposed. By applying a 30 mW heating power, a temperature gradient along antennas is generated and beam steering of 2.3° is achieved, satisfying applications such as long-range collision avoidance for autonomous driving.

Improved performances of optical phased arrays have been achieved assisted by graphene nanoheaters and air trenches.     

3D Pd/Co core–shell nanoneedle arrays as a high-performance cathode catalyst layer for AAEMFCs

A novel cathode architecture using vertically aligned Co nanoneedle arrays as an ordered support for application in alkaline anion-exchange membrane fuel cells (AAEMFCs) has been developed. The Co nanoneedle arrays were directly grown on a stainless steel sheet via a hydrothermal reaction and then a Pd layer was deposited on the surface of the Co nanoneedle arrays using a vacuum sputter-deposition method to form Pd/Co nanoneedle arrays. After transferring the Pd/Co nanoneedle arrays to an AAEM, a cathode catalyst layer was formed. Without the use of an alkaline ionomer, the AAEMFC with the prepared cathode catalyst layer showed an enhanced performance with ultra-low Pd loading of down to 33.5 μg cm⁻², which is much higher than the conventionally used cathode electrode with a Pt loading of 100 μg cm⁻². This is the first report where three-dimensional Co nanoneedle arrays have been used as the cathode support in an AAEMFC, which is able to deliver a higher power density without an alkaline ionomer than that of conventional membrane electrode assembly (MEA).

A novel ordered Pd/Co nanoneedle array was used as a cathode in an AAEMFC and delivered a higher power density than that of conventional MEA.     

Fatigue endurance enhancement of Sn-doped Pb(Lu1/2Nb1/2)O3–PbTiO3 ceramics

Several mechanisms and methods have been proposed to study the nature of electric fatigue in ferroelectric materials with perovskite structure, including defect agglomeration, field screening and the reorientation of defect dipoles. To ascertain the effect of defect, defect dipoles in particular on the fatigue behavior in perovskite ferroelectrics, 0.51Pb(Lu_1/2Nb_1/2)O₃–0.49PbTi_1−xSn_xO₃ ferroelectric ceramics were fabricated in this work. It is found that the fatigue endurance has been enhanced after Sn-doping. An abnormal strong self-rejuvenation of polarization was also detected for un-poled and un-aged samples resulting from the reorientation of defect dipoles. The defect dipoles were determined by the confirmed change of the valence of Sn ions and the appearance of oxygen vacancies. The reorientation was also confirmed by the internal bias of P–E hysteresis loops during the fatigue process. With more Sn doped into the matrix, the symmetry changed from a coexistence of rhombohedral and tetragonal phase to a rhombohedral phase. The remnant polarization decreased, while the coercive field first decreased then increased as x increased, which resulted from the composition variance and the effect of defect dipoles. It indicates that the defect dipoles play an important role in the electric fatigue behavior of Sn-doping PLN–PT ceramics.

Fatigue endurance has been enhanced of Sn-doped Pb(Lu_1/2Nb_1/2)O₃–PbTiO₃ ceramics, exhibiting an abnormal self-rejuvenation of remnant polarization.