A rapid analysis of antioxidants in Sanghuangporus baumii by online extraction-HPLC-ABTS

In the present study, a simple and efficient approach based on the online extraction-high performance liquid chromatography coupled with ABTS antioxidant assay (OLE-HPLC-ABTS) was established to quickly and directly analyze the antioxidants in S. baumii. Through this system, the HPLC mobile phase via a guard column packed with a S. baumii sample was used for online extraction (OLE). The separation was performed on an Agilent Poroshell EC-C18 column with a gradient elution using 0.1% formic acid (A) and 0.1% formic acid–acetonitrile (B) as mobile phase systems and detected at a wavelength of 254 nm. Then, the separated compounds were reacted with the antioxidant solution (ABTS), and the response was recorded at a wavelength of 400 nm. The developed analytical method was successfully applied to S. baumii samples, and eight antioxidants were identified. The established system integrated the online extraction, separation and online antioxidant detection, which is rapid, efficient, and suitable for the rapid screening of antioxidant compounds from solid sample mixtures.

Rapid discovery of antioxidants from natural product by online OLE-HPLC-ABTS.     

Enzymatic dephosphorylation-triggered self-assembly of DNA amphiphiles

In this work, phosphorylated lipid-conjugated oligonucleotide (DNA-lipid-P) has been synthesized to develop an enzyme-responsive self-assembly of DNA amphiphiles based on dephosphorylation-induced increase of hydrophobicity. Since elevated ALP level is a critical index in some diseases, ALP-triggered self-assembly of DNA amphiphiles shows promise in disease diagnosis and cancer treatment.

Enzymatic dephosphorylation-triggered self-assembly of DNA amphiphiles is developed by integrating enzymatic dephosphorylation-induced increase of hydrophobicity and intermolecular aggregation of lipid-conjugated oligonucleotides.

Lipid-conjugated oligonucleotides are DNA amphiphiles that can self-assemble into lipid-based DNA micelles. As such, lipid tails act as a hydrophobic core and DNA act as a hydrophilic corona.^1–3 Because of their advantages of facile preparation, programmable design, small size (<100 nm) and biocompatibility, lipid-based DNA micelles show potential in the imaging of intracellular targets (mRNA and small molecules) and drug delivery.^4–6To further improve their potential for better drug delivery and disease diagnosis, stimuli-responsive control of the change of morphology or assembly/disassembly of DNA micelles has attracted enormous attention in practical applications. It was reported that photo-irradiation and nucleic acid hybridization have been used to trigger assembly/disassembly or the change of morphology of DNA micelles.^7–10 For example, Jin et al. developed stability-tunable DNA micelles by using photo-controllable dissociation of an intermolecular G-quadruplex.⁷ The intermolecular parallel G-quadruplexes were introduced into lipid-based DNA micelles to lock the whole structure, resulting in enhanced structural stability against disruption by serum albumin. However, photo-controlled release of complementary DNA blocks the formation of G-quadruplexes and thus leads to the dissociation of micelles by the existence of serum albumin. In addition, Chien et al. reported stimuli-responsive programmable shape-shifting DNA micelles by controlling geometric structure and electrostatics.⁸ DNA hybridization and dissociation change the geometric structure and electrostatics of hydrophilic moiety, leads to the conversion of DNA assemblies between spherical and cylindrical structures.In spite of these advances, enzyme-responsive regulation of self-assembly of DNA amphiphiles has scarcely reported. Since the hydrophobicity of lipid tail plays a critical role in the aggregation of DNA micelles, we speculated that the self-assembly of lipid-conjugated oligonucleotides can be regulated by controlling the hydrophobicity of lipid tails. Carry this idea forward, we noted that ALP is a hydrolase that removes phosphate groups from nucleic acids or proteins.^11,12 Remarkably, ALP has been widely employed to convert hydrophilic phosphorylated small molecules to hydrophobic dephosphorylated products for molecular imaging, disease diagnosis and cancer therapy.^13–16 Herein, therefore, enzymatic dephosphorylation-triggered self-assembly of DNA amphiphile is reported. As shown in Fig. 1, DNA-lipid-P is composed of four segments: DNA, linker, lipid and phosphate groups. Four negatively charged phosphate groups at lipid terminus decrease their hydrophobicity. Therefore, DNA-lipid-P exhibits weak self-assembly. However, ALP converts DNA-lipid-P to DNA-lipid by removing phosphate groups. The newly generated DNA-lipid shows greater hydrophobicity compared to DNA-lipid-P thus enables self-aggregation in aqueous solution.Open in a separate windowFig. 1Schematic illustration of enzymatic dephosphorylation-triggered self-assembly of DNA amphiphile.Lipid-conjugated oligonucleotides are amphiphiles which compose of two segments: hydrophobic lipid tails and hydrophilic oligonucleotides. Generally, lipid-conjugated oligonucleotides can self-assemble into aggregated DNA nanostructures, for example, DNA micelles, in aqueous solution by intermolecular hydrophobic interaction. To investigate whether the hydrophobicity affects the self-assembly of lipid-conjugated oligonucleotides, a series of lipid phosphoramidites with different length of alkyl chains (six, nine, twelve and fifteen) at the lipid tail were conjugated with oligonucleotide on a DNA synthesizer. The obtained lipid-conjugated oligonucleotides named as C6-DNA, C9-DNA, C12-DNA and C15-DNA, respectively (Fig. 2a). High-performance liquid chromatography (HPLC) is a universal tool to assess the hydrophobicity of DNA by comparing their retention times. Greater retention time indicates the stronger hydrophobicity. As shown in Table S2,^† the retention time of DNA, C6-DNA, C9-DNA, C12-DNA and C15-DNA is 10.0, 19.1, 23.8, 27.3 and 30.8 minutes, respectively, suggesting that DNA with longer alkyl chains has stronger hydrophobicity. The result is consistent with the previous report.¹⁷ Next, the self-assembly of these lipid-conjugated oligonucleotides was investigated by agarose gel electrophoresis and dynamic light scattering (DLS) assays. As shown in Fig. 2b, only C15-DNA shows a tailed nucleic acids band which is belongs to the self-assembled nanostructure. In addition, results of DLS assays exhibit that the particle size of 10 μM C6-DNA, C9-DNA, C12-DNA and C15-DNA in buffer solution is 2.7 nm, 4.2 nm, 6.5 nm and 28.2 nm, respectively (Fig. 2c). Besides, the morphology of self-assembled C15-DNA micelles was visualized with atomic force microscopy (AFM) and the result shows the spherical nanostructure with diameter of 36.8 ± 6.1 nm (Fig. S7^†). Both evidences support the self-assembly of C15-DNA in buffer solution. In another word, the self-assembly of lipid-conjugated oligonucleotides into DNA micelles is hydrophobicity-dependent. A greater hydrophobicity indicates a stronger tendency of aggregation.Open in a separate windowFig. 2Hydrophobicity-dependent self-assembly of lipid-conjugated oligonucleotides. (a) Chemical structures of lipid-conjugated oligonucleotides with different length of alkyl chains at the terminus of lipid tail. (b) 1% agarose gel electrophoresis analysis of 1 μM TAMRA-labeled C6-DNA, C9-DNA, C12-DNA and C15-DNA. C15-DNA shows a tailed band in agarose gel which can be attributed to the formation of aggregated micellar nanostructure. (c) DLS size analysis of C6-DNA, C9-DNA, C12-DNA and C15-DNA in buffer solution. The average size of C6-DNA, C9-DNA, C12-DNA and C15-DNA in buffer solution is 2.7 nm, 4.2 nm, 6.5 nm and 28.2 nm, respectively.Next, we further synthesize DNA-lipid-P by solid-phase synthesis and phosphoramidite chemistry. In a previous literature, we developed a novel lipid phosphoramidite in which two DMT-protected hydroxyl groups was modified at the terminus of lipid tails thus enables further chemical phosphorylation during DNA synthesis.¹⁷ As shown in Fig. 3, linker and lipid phosphoramidites were successively conjugated at the 5′-terminus of DNA, followed by coupling with chemical phosphorylation reagent. After deprotection and purification, DNA-lipid-P was obtained and characterized by mass spectrum. As shown in Fig. S8,^† the calculated molecular weight of DNA-lipid-P is 7789.8 Da, and the observed molecular weight is 7792.4 Da. The mass error is 2.6 Da (0.03%) which is within the mass error tolerance (0.03%), suggesting the successful synthesis of DNA-lipid-P. As such, DNA-lipid was also successfully synthesized with high purity (>98%) (Fig. S9^†).Open in a separate windowFig. 3Solid-phase synthesis route of DNA-lipid-P.Having confirmed the successful synthesis of DNA-lipid-P, we further investigate the self-assembly of DNA-lipid and DNA-lipid-P in buffer solution. Nile red, a fluorescent dye that exhibits significant fluorescence in hydrophobic media, but negligible emission in aqueous solution, was used to determine the encapsulation of guest molecules to further assess the formation of the micellar structure.^7,18 Nile red (1 μM) were incubated with various concentrations of DNA, DNA-lipid or DNA-lipid-P and the corresponding fluorescence spectroscopies were recorded. As shown in Fig. 4, both DNA (Fig. 4b) and DNA-lipid-P (Fig. 4c) show weaker fluorescence emission at 630 nm, even the concentration was upper to 10 μM. However, DNA-lipid (Fig. 4d) exhibits a bright fluorescence emission at 630 nm, suggesting the formation of hydrophobic core. After calculation, the critical micelle concentration (CMC) of DNA-lipid is 0.36 μM (Fig. 4e). The remarkable difference of CMC between DNA-lipid and DNA-lipid-P indicates that enzymatic conversion of DNA-lipid-P to DNA-lipid could trigger the spontaneous intermolecular aggregation.Open in a separate windowFig. 4Characterizations of self-assembly of DNA-lipid-P and DNA-lipid. (a) The chemical structures of DNA-lipid and DNA-lipid-P. Fluorescence spectroscopies of Nile red-encapsulated DNA (b), DNA-lipid-P (c) and DNA-lipid (d) in buffer solution. The concentration of Nile red is 1 μM. (e) Fluorescence intensity of Nile red-encapsulated DNA, DNA-lipid-P and DNA-lipid at 630 nm. The CMC of DNA-lipid is 0.36 μM, and the CMC of DNA-lipid-P is larger than 10 μM.Next, ALP was used to convert DNA-lipid-P to DNA-lipid (Fig. 5a). As shown in Fig. 5b (black and red lines), the retention time of DNA-lipid and DNA-lipid-P is 26.5 and 20.9 minutes, respectively, indicates that the phosphorylation of lipid tail indeed decreases the hydrophobicity of lipid-conjugated oligonucleotides. Then, ALP was incubated with DNA-lipid-P (10 μM) at 37 °C for ten minutes and then 75 °C for five minutes to deactivate ALP, followed by subjected to fluorescence measurements. As shown in Fig. 5b (pink line), after the treatment of ALP (2 U), the DNA peak of DNA-lipid-P at 20.9 minutes disappeared; instead, a new DNA peak at 26.5 minutes was observed. Mass spectrum analysis indicates that the molecular weight of newly generated DNA peak is 7474.2 Da, which is consistent with the calculated molecular weight of DNA-lipid (7470.9 Da) (Fig. S10^†). In a word, ALP enables enzymatic dephosphorylation of DNA-lipid-P; and the generated DNA-lipid has greater hydrophobicity than DNA-lipid-P thus facilitates the controllable self-assembly into DNA micelles.Open in a separate windowFig. 5Enzymatic dephosphorylation of DNA-lipid-P. (a) Schematic of ALP-induced conversion of DNA-lipid-P to DNA-lipid. (b) HPLC chromatograms of DNA-lipid-P (black line), DNA-lipid (red line), and DNA-lipid-P treated with ALP (0.1 U (blue line), 1 U (green line) and 2 U (pink line)) in buffer solution.Encouraged by the ALP-induced dephosphorylation of DNA-lipid-P to DNA-lipid, we further assess whether ALP enables activatable self-assembly of DNA-lipid-P. As shown in Fig. 6a, DNA-lipid-P (1 μM) shows weak self-assembly in buffer solution. However, DNA-lipid (1 μM) exhibits obvious aggregation band in gel electrophoresis assay. After incubation with ALP (1 U), DNA-lipid-P + ALP group also shows tailed band which suggests the formation of aggregated nanostructures. In addition, results of Nile red-encapsulated fluorescence experiments also support the conclusion of ALP-activate self-assembly of DNA-lipid-P (Fig. 6b). Therefore, ALP-induced enzymatic dephosphorylation triggers the self-assembly of DNA-lipid-P.Open in a separate windowFig. 6Enzymatic dephosphorylation-triggered self-assembly of DNA-lipid-P. (a) 1% agarose gel electrophoresis analysis of DNA-lipid-P (lane 1), DNA-lipid (lane 2) and DNA-lipid-P treated with ALP (1 U) (lane 3). (b) Fluorescence spectroscopies of Nile red-encapsulated DNA-lipid, DNA-lipid (ALP), DNA-lipid-P or DNA-lipid-P (ALP).In summary, enzymatic dephosphorylation-triggered self-assembly of DNA amphiphile is developed by integrating enzymatic dephosphorylation-induced increase of hydrophobicity and intermolecular aggregation of lipid-conjugated oligonucleotides. The strategy may also suitable for many other amphiphiles. Since elevated ALP level is a critical index in some diseases and even cancers, we believe that ALP-triggered self-assembly of DNA-lipid-P shows potential in disease diagnosis and cancer therapy.     

Structural insights into targeting of the colchicine binding site by ELR510444 and parbendazole to achieve rational drug design

Microtubules consisting of α- and β-tubulin heterodimers have proven to be an efficient drug target for cancer therapy. A broad range of agents, including ELR510444 and parbendazole, can bind to tubulin and interfere with microtubule assembly. ELR510444 and parbendazole are colchicine binding site inhibitors with antiproliferative activities. However, the lack of structural information on the tubulin–ELR510444/parbendazole complex has hindered the design and development of more potent drugs with similar scaffolds. Therefore, we report the crystal structures of tubulin complexed with ELR510444 at a resolution of 3.1 Å and with parbendazole at 2.4 Å. The structure of these complexes revealed the intermolecular interactions between the two colchicine binding site inhibitors and tubulin, thus providing a rationale for the development of novel benzsulfamide and benzimidazole derivatives targeting the colchicine binding site.

Crystal structures of tubulin complexed with ELR510444 and parbendazole facilitate the design of novel colchicine binding site inhibitors.     

Self-supported Cu3P nanowire electrode as an efficient electrocatalyst for the oxygen evolution reaction

Hydrogen is an ideal energy carrier due to its abundant reserves and high energy density. Electrolyzing water is one of the carbon free technologies for hydrogen production, which is limited by the sluggish kinetics of the half reaction of the anode – the oxygen evolution reaction (OER). In this study, a self-supported Cu₃P nanowire (Cu₃P NWs/CF) electrode is prepared by electrodeposition of a Cu(OH)₂ nanowire precursor on conductive Cu foam (Cu(OH)₂ NWs/CF) with a subsequent phosphating procedure under a N₂ atmosphere. When used as an OER working electrode in 1.0 M KOH solution at room temperature, Cu₃P NWs/CF exhibits excellent catalytic performance with an overpotential of 327 mV that delivers a current density of 20 mA cm⁻². Notably, it can run stably for 22 h at a current density of 20 mA cm⁻² without obvious performance degradation. This highly efficient and stable OER catalytic performance is mainly attributed to the unique nanostructure and stable electrode construction. Interestingly, this synthesis strategy has been proved to be feasible to prepare large-area working electrodes (e.g. 40 cm⁻²) with unique nanowire structure. Therefore, this work has provided a good paradigm for the mass fabrication of self-supporting non-noble metal OER catalysts and effectively promoted the reaction kinetics of the anode of the electrolyzing water reaction.

We prepared Cu₃P nanowires via a simple two-step method and Cu(OH)₂ NWs/CF was converted to Cu₃P/NWs after a phosphating process. The prepared Cu₃P NWs/CF electrode shows high efficiency and excellent stability to OER in alkaline medium.     

对腹膜透析中腹膜血管生成的认识

长期腹膜透析会引起腹膜血管生成,对腹膜功能有重大的影响。本文就尿毒症腹膜透析患者腹膜血管生成的原因,血管形成的病理生理过程及腹膜血管新生对腹膜功能的影响做一综述。     

Gasdermin D inhibition confers antineutrophil-mediated cardioprotection in acute myocardial infarction

Acute myocardial infarction (AMI) induces blood leukocytosis, which correlates inversely with patient survival. The molecular mechanisms leading to leukocytosis in the infarcted heart remain poorly understood. Using an AMI mouse model, we identified gasdermin D (GSDMD) in activated leukocytes early in AMI. We demonstrated that GSDMD is required for enhanced early mobilization of neutrophils to the infarcted heart. Loss of GSDMD resulted in attenuated IL-1β release from neutrophils and subsequent decreased neutrophils and monocytes in the infarcted heart. Knockout of GSDMD in mice significantly reduced infarct size, improved cardiac function, and increased post-AMI survival. Through a series of bone marrow transplantation studies and leukocyte depletion experiments, we further clarified that excessive bone marrow–derived and GSDMD-dependent early neutrophil production and mobilization (24 hours after AMI) contributed to the detrimental immunopathology after AMI. Pharmacological inhibition of GSDMD also conferred cardioprotection after AMI through a reduction in scar size and enhancement of heart function. Our study provides mechanistic insights into molecular regulation of neutrophil generation and mobilization after AMI, and supports GSDMD as a new target for improved ventricular remodeling and reduced heart failure after AMI.     

不同氮素形态和浓度水平对浙贝母产量和品质的影响

目的:研究不同氮素形态配施对浙贝母产量和品质的影响,为浙贝母氮肥的科学施用及重庆地区的迁地引种提供依据。方法:采用盆栽试验法,研究了2种氮素形态的5种浓度水平即硝态氮(NO_3~--N)-铵态氮(NH_4~+-N)为15∶0(N1),12∶3(N2),7. 5∶7. 5(N3),3∶12(N4),0∶15(N5)处理下,引种栽培的浙贝母生长及生理生化、土壤因子、生物碱含量及产量的变化。结果:与不施氮(CK)处理组相比,不同氮素营养配施对浙贝母生长和品质均有显著提高,相互间存在差异。其中,随着铵态氮浓度的增加:①在硝态氮-铵态氮比为3∶12时,株高、超氧化物歧化酶(SOD)活性达到最大,较CK组分别增加了9. 27%,206. 62%;②在硝态氮-铵态氮比为0∶15时,叶长,叶宽,茎粗,叶绿素a,叶绿素b,叶绿素总量,速效磷含量,有机质含量、总生物碱含量/产量达到最大,分别高于CK处理组14. 02%,16. 44%,13. 68%,40. 75%,45. 31%,41. 72%,77. 70%,14. 70%,24. 61%/47. 39%;随着硝态氮浓度的增加;③在硝态氮-铵态氮比为7. 5∶7. 5时,叶形指数、可溶性蛋白含量、贝母辛含量/产量、贝母素乙产量、鳞茎干重均达到最大,分别高于CK处理组2. 54%,5. 92%,21. 76%/54. 55%,60. 61%,26. 93%;④在硝态氮-铵态氮比为12∶3时,类胡萝卜素含量、色素含量、过氧化物酶(POD)活性、过氧化氢酶(CAT)活性、贝母素甲含量/产量、贝母素乙含量、贝母素(甲+乙)含量/产量、贝母素(甲+乙)+贝母辛含量/产量、鳞茎湿重均达到最大,分别高于CK处理组45. 39%,45. 31%,271. 38%,67. 45%,39. 82%/64. 87%,36. 01%,38. 90%/63. 80%,37. 03%/61. 57%,20. 29%。结论:较高比例的铵态氮利于浙贝母的生长;而较高比例的硝态氮利于浙贝母鳞茎的生长以及代谢产物生物碱的积累。氮素配比施用(硝态氮-铵态氮比为12:3)较单一形态氮素更有利于提高浙贝母的产量和品质。