Retransplantation for severe accelerated coronary artery disease in heart transplant recipients

Development of accelerated coronary artery disease (CAD) in the cardiac allograft is one of the major causes of late graft failure in heart transplant recipients. At the Stanford University Medical Center 356 heart transplant procedures were performed in 329 patients by the end of January 1985. Eighty-nine of these patients developed evidence of transplant CAD. Twenty retransplant procedures, including 2 third transplants, were performed in 19 of the 89 patients because of transplant CAD. The graft survival rates after the second transplant were 55%, 25% and 10% after 1, 2 and 5 years, respectively. Nine of these retransplant patients currently survive, the longest for 5.5 years. To examine potential risk factors for development of severe transplant CAD, these 20 retransplant procedures were compared with 113 transplant recipients who had no evidence of transplant CAD on annual coronary arteriograms. An excess of rejection episodes (3 +/- 2 vs 2 +/- 1 episodes/patient, p = 0.02), elevated total cholesterol (266 +/- 78 vs 225 +/- 47 mg/dl, p = 0.002) and higher low-density lipoprotein levels (176 +/- 88 vs 137 +/- 46 mg/dl, p = 0.009) were noted in the transplant CAD retransplant group. Five of 11 retransplant recipients who survived greater than 1 year again developed transplant CAD. Characteristic morphologic features and rapid progression of CAD in the second graft were similar to those in the primary graft.(ABSTRACT TRUNCATED AT 250 WORDS)     

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).     

Three-dimensional honeycomb-like porous carbon derived from corncob for the removal of heavy metals from water by capacitive deionization

In this study, porous carbon (3DHPC) with a 3D honeycomb-like structure was synthesized from waste biomass corncob via hydrothermal carbonization coupled with KOH activation and investigated as a capacitive deionization (CDI) electrode material. The obtained 3DHPC possesses a hierarchal macroporous and mesoporous structure, and a large accessible specific surface area (952 m² g⁻¹). Electrochemical tests showed that the 3DHPC electrode exhibited a specific capacitance of 452 F g⁻¹ and good electric conductivity. Moreover, the feasibility of electrosorptive removal of chromium(vi) from an aqueous solution using the 3DHPC electrode was demonstrated. When 1.0 V was applied to a solution containing 30 mg L⁻¹ chromium(vi), the 3DHPC electrode exhibited a higher removal efficiency of 91.58% compared with that in the open circuit condition. This enhanced adsorption results from the improved affinity between chromium(vi) and the electrode under electrochemical assistance involving a non-faradic process. Consequently, the 3DHPC electrode with typical double-layer capacitor behavior is demonstrated to be a favorable electrode material for capacitive deionization.

A porous carbon electrode with a 3D honeycomb-like structure demonstrates a high removal efficiency for the removal of chromium(vi) from water.     

The effect of beta-sitosterol and its derivatives on depression by the modification of 5-HT,DA and GABA-ergic systems in mice

Beta-sitosterol belongs to the group of phytosterols, which are active trace components existing in natural plants, known as the “key of life”, and have a steroid nucleus structure similar to cholesterol. Due to the insolubility issue of beta-sitosterol, most pharmacological studies and clinical applications are limited. Therefore, the modification of beta-sitosterol into its derivatives to enhance its pharmacologic activity is viable. In this study, 4 kinds of new beta-sitosterol derivative were obtained by an esterification reaction with beta-sitosterol, organic acids, EDCI and DMAP in dichloromethane. The chemical structures were defined by IR and NMR. Beta-sitosterol and its derivatives were used to carry out antidepressant research in the tail suspension test (TST) and the forced swimming test (FST) in mice. Additionally, the roles of different parts of the central nervous system (CNS) in the antidepressant-like effect of Sit-S, which is one of the beta-sitosterol derivatives, were also investigated. The results showed that the derivatives exhibited a stronger antidepressant activity than beta-sitosterol. Among the derivatives, administration of Sit-S (4 mg kg⁻¹) gave the lowest immobility time in the TST, demonstrating that Sit-S exhibited the strongest antidepressant-like activity. The study into the roles of different parts of the CNS in the antidepressant-like effect of Sit-S showed that agomelatine (40 mg kg⁻¹), haloperidol (0.2 mg kg⁻¹) and bicuculline (4 mg kg⁻¹) reversed the antidepressant effect of Sit-S (4 mg kg⁻¹). This study confirmed the conclusions that beta-sitosterol derivatives broaden the pharmacological effects of beta-sitosterol, Sit-S (4 mg kg⁻¹) exhibits antidepressant-like effects, and this antidepressant-like effect on male adult mice is mediated by the 5-HT, DA and GABA-ergic systems.

Beta-sitosterol and its derivatives exhibit antidepressant-like activity mediated by the modification of 5-HT, DA and GABA-ergic systems.     

Physiologically stable F127-GO supramolecular hydrogel with sustained drug release characteristic for chemotherapy and photothermal therapy

A synthetic method for preparing a Pluronic F127 (F127)-stabilized graphene (GO) supramolecular hydrogel as a safe nanovehicle for combination treatment has been studied. Doxorubicin (DOX) as a model drug is non-covalently bound on the great surface area of GO due to strong π–π interaction, hydrophobic interaction, and the strongest hydrogen bonding. In vitro drug release experiments revealed that this F127-stabilized GO supramolecular hydrogel has a sustained drug release characteristic. Furthermore, the supramolecular hydrogel showed better in vitro antitumor ability under NIR (near infrared) laser irradiation because of the excellent photothermal effect of GO. Moreover, we evaluated its antitumor ability in vivo and the results show that the hydrogel system can also markedly inhibit the growth of a tumor when administered individually, especially under laser irradiation. All these findings make the supramolecular hydrogel system promising for combination therapy with good bioavailability and minimal side effects.

The F127-GO-DOX supramolecular hydrogel system with sustained drug release characteristic for chemotherapy and photothermal therapy.     

N-Acetyl-l-leucine-polyethylenimine-mediated miR-34a delivery improves osteogenesis and bone formation in vitro and in vivo

Oral bone defects are difficult to treat. Recently, endogenous miR-34a was shown to be involved in bone anabolism. Clinical application of such microRNAs requires the inherent instability of microRNAs to be overcome by an efficient delivery system. In this study, we employed N-acetyl-l-leucine-modified polyethylenimine (N-Ac-l-Leu-PEI) as an miR-34a carrier and evaluated its delivery ability, transfection efficiency, cytotoxicity and whether it enhanced osteogenic differentiation and bone formation in vitro and in vivo. Stable N-Ac-l-Leu-PEI/miR-34a nanocomplexes were synthesized at a mass ratio of 4 and had a small size (190.34 nm), a low zeta potential (21.1 mV), a high transfection efficiency (69.39%) and no cytotoxicity in MG63 cells. N-Ac-l-Leu-PEI-mediated miR-34a delivery in vitro promoted ALP activity and expression of osteogenic differentiation markers, Runx2, SP7 and ColI to higher levels than those produced by Lipofectamine 2000-mediated delivery. N-Ac-l-Leu-PEI also achieved delivery of miR-34a in vivo to a local cranial bone defect area with miR-34a retaining the ability to initiate significant new bone formation 12 weeks post-implantation. This demonstrates the potential for N-Ac-l-Leu-PEI as a gene therapy vehicle for the regeneration of bone defects.

We employ N-acetyl-l-leucine-modified polyethylenimine as an miR-34a carrier and evaluate its delivery ability, transfection efficiency, cytotoxicity and whether it enhances osteogenic differentiation and bone formation in vitro and in vivo.     

Electrolytic extraction of dysprosium and thermodynamic evaluation of Cu–Dy intermetallic compound in eutectic LiCl–KCl

The electrochemical reduction of dysprosium(iii) was studied on W and Cu electrodes in eutectic LiCl–KCl by transient electrochemical methods. Cyclic voltammogram and current reversal chronopotentiogram results demonstrated that dysprosium(iii) was directly reduced to dysprosium (0) on the W electrode through a single-step process with the transfer of three electrons. Electrochemical measurements on the Cu electrode showed that different Cu–Dy intermetallics are formed. Moreover, the thermodynamic properties of Cu–Dy intermetallic compounds were estimated by open circuit chronopotentiometry in a temperature range of 773–863 K. Using the linear polarization method, the exchange current density (j₀) of dysprosium in eutectic LiCl–KCl on the Cu electrode was estimated, and the temperature dependence of j₀ was studied to estimate the activation energies associated with Dy(iii)/Cu₅Dy and Dy(iii)/Cu_9/2Dy couples. In addition, potentiostatic electrolysis was conducted to extract dysprosium on the Cu electrode, and five Cu–Dy intermetallic compounds, CuDy, Cu₂Dy, Cu_9/2Dy, Cu₅Dy and Cu_0.99Dy_0.01 were identified by X-ray diffraction, scanning electron microscopy and energy dispersive spectrometry. Meanwhile, the change of dysprosium(iii) concentration was monitored using inductively coupled plasma-atomic emission spectrometry, and the maximum extraction efficiency of dysprosium was found to reach 99.2%.

The electrochemical reduction of dysprosium(iii) was studied on W and Cu electrodes in eutectic LiCl–KCl by transient electrochemical methods.     

Direct automatic determination of the methanol content in red wines based on the temperature effect of the KMnO4/K2S2O5/fuchsin sodium sulfite reaction system

The standard method for methanol assay in wine is based on a methanol/KMnO₄/H₂C₂O₄/fuchsin sodium sulfite (FSS) reaction system. However, it is difficult to control the degree of colour and the temperature of the reaction product in this assay, and its repeatability is also poor due to the generation of CO and CO₂ in the reaction. Therefore, to solve these problems, potassium metabisulfite was selected to replace H₂C₂O₄, and an automatic analysis method was developed which can realize rapid and accurate determination of methanol and can be used to make an online analyzer. It was discovered that the reactions of methanol/KMnO₄ and acetaldehyde/FSS are exothermic, while the reactions of methanol/KMnO₄ and formaldehyde/FSS are endothermic. Consequently, based on the temperature effect, not only was the interference of ethanol eliminated in detecting methanol in wines, the purpose of the research was achieved to directly and accurately determine methanol without sample pretreatment. By optimizing the system, the obtained conditions for determining methanol in wines were as follows: 20 g L⁻¹ concentration for KMnO₄; 3 g L⁻¹ concentration for FSS; 40 cm length for the first reaction coil (RC₁); 100 cm length for RC₂; 700 cm (I.D.: 0.8 mm) length for RC₃; 50 °C for RC₃; about 20 °C for RC₁ and RC₂; 330 μL for the sample volume. The method showed a linear response in the range 25–1000 mg L⁻¹, with a 0.6% RSD, 8.8 mg L⁻¹ detection limit and 25 samples per h, and was successfully used for testing representative wine samples. It also obtained better accuracy than previous methods. Due to its superiority in automated operation, reproducibility, analysis speed and test cost, this method and system can serve as a supplementary standard for methanol assay, and for the quality control of the winemaking process and the final wine-product, as well as for low-alcohol drinks.

We found that methanol/KMnO₄ and acetaldehyde/FSS are exothermic, formaldehyde/FSS is endothermic, so based on the temperature effect, eliminated ethanol interference in detecting methanol in wines, also realized automatic analysis for methanol without sample pretreatment.     

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.     

Membrane adaptation limitations in Enterococcus faecalis underlie sensitivity and the inability to develop significant resistance to conjugated oligoelectrolytes

The growing problem of antibiotic resistant bacteria, along with a dearth of new antibiotics, has redirected attention to the search for alternative antimicrobial agents. Conjugated oligoelectrolytes (COEs) are an emerging class of antimicrobial agents which insert into bacterial cell membranes and are inhibitory against a range of Gram-positive and Gram-negative bacteria. In this study, the extent of COE resistance that Enterococcus faecalis could achieve was studied. Enterococci are able to grow in hostile environments and develop resistance to membrane targeting antibiotics such as daptomycin in clinical settings. Herein we expand our knowledge of the antimicrobial mechanism of action of COEs by developing COE-resistant strains of E. faecalis OG1RF. Evolution studies yielded strains with a moderate 4–16 fold increase in antimicrobial resistance relative to the wild type. The resistant isolates accumulated agent-specific mutations associated with the liaFSR operon, which is a cell envelope-associated stress-response sensing and regulating system. The COE resistant isolates displayed significantly altered membrane fatty acid composition. Subsequent, exogenous supplementation with single fatty acids, which were chosen based on those dominating the fatty acid profiles of the mutants, increased resistance of the wild-type E. faecalis to COEs. In combination, genetic, fatty acid, and uptake studies support the hypothesis that COEs function through insertion into and disruption of membranes and that the mechanism by which this occurs is specific to the disrupting agent. These results were validated by a series of biophysical experiments showing the tendency of COEs to accumulate in and perturb adapted membrane extracts. Collectively, the data support that COEs are promising antimicrobial agents for targeting E. faecalis, and that there is a high barrier to the emergence of severely resistant strains constrained by biological limits of membrane remodeling that can occur in E. faecalis.

COEs are emerging antimicrobials to combat drug resistant infections and to which bacteria develop only limited resistance.