The physicochemical properties of a room-temperature liquidus binary ionic liquid mixture of [HNMP][CH3SO3]/[Bmim]Cl and its application for fructose conversion to 5-hydroxymethylfurfural

Via heating first and then cooling, binary ionic liquid (IL) mixture of N-methyl-2-pyrrolidonium methylsulfonate ([HNMP][CH₃SO₃]) and 1-butyl-3-methylimidazolium chloride ([Bmim]Cl) could form a liquid at room temperature. The glass-transition temperature (T_g) characterized by DSC depends on its composition with T_g being as low as −63 °C. The physicochemical properties of the binary IL mixtures also vary with the composition. With the increase of the mole fraction of [Bmim]Cl, the hydrogen-bond accepting ability (β) of the binary IL mixture increases, but the hydrogen-bond donating ability (α) deceases. In this binary IL mixture, fructose could be effectively converted into 5-hydroxymethylfurfural (HMF) at room temperature. The HMF yields at a given time are found to be well correlated with the physicochemical properties of the binary mixture, especially the α and β values. Under specified conditions, the present IL mixture as medium for fructose dehydration into HMF is comparable to the medium formed by ILs and alcohol, where the alcohols have negative effect on the HMF formation.

A liquidus mixture of [HNMP][CH₃SO₃]/[Bmim]Cl has been characterized and tried as medium for room-temperature conversion of fructose into HMF.     

Irregular solution thermodynamics of wood pulp in the superbase ionic liquid [m-TBDH][AcO]

Knowledge of solution thermodynamics is fundamental for solution control and solvent selection processes. Herein, experimentally determined thermodynamic quantities for solutions of wood pulp (hardwood dissolving pulp, i.e. cellulose) in [m-TBDH][AcO] are presented. Model-free activities (a_i,j) and associated mass fraction (w_i,j) activity coefficients (Ω_i,j), are determined to quantify inherent solution non-ideality. Access to the Gibbs energy of mixing, G_mix, in combination with associated partial molar thermodynamic quantities, reveal strong enthalpically favourable (exothermic) interactions due to solvent-j and solute-i contact-encounters. Onset of an entropy driven phase instability appears at increased temperatures as excess entropic contributions dominate solvation character of the irregular solutions formed.

Thermochemical analysis of cellulose dissolution character in the superbase containing protic ionic liquid [m-TBDH][AcO] reveals lower critical solution temperature (LCST) behaviour.

Investigations concerning dissolution of carbon-neutral biomass components in varieties of ionic liquids (ILs) have increasingly populated the literature since the turn of the century.¹ From this renewed interest in the discovery and development of powerful new solvents for difficult to dissolve polymeric bio-solutes, protic “superbase” ILs have emerged as promising candidates for cellulose dissolution and upstream processing related to a variety of materials.^2–8 To date, focus in the literature has largely been bounded by efforts to identify liquid salts with qualitative dissolution utility for biomass components, among various contributions detailing new developments in synthetic carbohydrate derivatisation chemistry.^9,10 Much less attention has been given towards gaining a detailed understanding of the energetics governing solvent–solute contact-encounters in such mixtures, using robust thermodynamic methods.¹¹Knowledge gained from such approaches reveal available composition- and temperature-dependent solvation character of both solvent-j and solute-i, which in turn provides the fundamental basis upon which the solvent selection process may be anchored, and furthermore, the means to identify a given solvent''s utility in terms of true task specificity. Herein, we report thermodynamic solution behaviour of cellulose dissolved in the superbase comprised IL, [m-TBDH][AcO] (7-methyl-1,5,7-triazabicyclo[4.4.0]dec-5-enium acetate). Employing a minimal experimental data set, we present the subsequent processing and interpretation of results obtained directly from fundamental thermodynamic relationships, thereby avoiding various well-known limitations introduced by numerous thermodynamic polymer models available (e.g. Flory–Huggins and associated theoretical developments).^12–17This investigation relies on the well-known colligative property defined by the freezing point depression of the solvent-j, in the presence of solute-i, at low mass fractions, w_i. In combination with the Gibbs–Duhem relation for a 2 component system, w_idln(a_i) + w_jdln(a_j) = 0, this permits the realisation of solute-i activities (a_i) based on those experimentally determined for the solvent-j (a_j). In essence, freezing point depressions for the binary solutions, recorded experimentally using modulated differential scanning calorimetry (MDSC), reveal changes to a_j using the variant of Kirchhoff''s law given in eqn (1)., imposed by the presence of solute-i (i.e. enocell-i)^‡, which perturbs the chemical potential of the solvent-j, [m-TBDH][AcO]-j, to a measurable extent.1Scaling of solvent activities, a_j, based on the depressed freezing temperature T, obtained from eqn (1)., to temperatures of interest, T′, was accomplished using the variant of the Gibbs–Helmholtz relation given in eqn (2),2where $\bar{H}$_j represents the relative partial molar enthalpy of solvent-j. The Gibbs–Duhem relation, utilised on the basis of the form given in eqn (3),3was subsequently employed to access, and verify, the thermodynamic consistency of desired excess thermodynamic quantities of solute-i.¹⁸Freezing point depressions recorded during the independently run triplicate MDSC experiments produced standard deviations for a given composition on the order of ± < 0.18 K. Solute-i activity coefficients, Ω_i, determined from experimentally recorded solvent-j activity coefficients, viaeqn (3), were subsequently employed to back-calculate experimentally determined values, again using eqn (3). The closeness of computed Ω_j, obtained using Ω_i, to the experimental values, serves as a consistency check, to validate and verify the Ω_i obtained. We observed very satisfactory consistency by this method, where differences between experimental and calculated Ω_j were generally ≪1% for dilute solutions, and up to ∼5% for certain w_j, at the highest temperature investigated (see the ESI^†).Selected thermodynamic results presented in ​and22 are discussed below in terms of the thermodynamic suitability of [m-TBDH][AcO] for the task of dissolving cellulose, based on the molar Gibbs mixing energies, in relation to associated partial molar enthalpic and entropic contributions.^§Experimentally determined thermodynamic quantities for binary mixtures of enocell-i and [m-TBDH][AcO]-j – variation of mass fraction, w_i, at constant temperature 360.15 K

Adsorption of arsenic from aqueous solution using a zero-valent iron material modified by the ionic liquid [Hmim]SbF6

The environmental and health impacts caused by arsenic (As) in wastewater make it necessary to carefully manage As wastes. In the present work, a composite of the ionic liquid [Hmim]SbF₆ and nano-iron (H/Fe) was used as an adsorbent to remove As(v) from aqueous solution. To better understand the removal effect of H/Fe on As(v) in aqueous solution, the reaction parameters of pH, reaction temperature, time and H/Fe dosage were systematically analyzed in detail. The results show that H/Fe has significant removal efficiency toward As(v), and that the adsorption of As(v) by 0.5 g H/Fe reaches its maximum adsorption capacity within 2 h. The adsorption of As(v) on H/Fe is a non-linear, time-varying process. The initial adsorption reaction is fast; however, unlike at the beginning, the later reaction involves sustained slow absorption, resulting in a distinct two-phase adsorption characteristic. Redox reaction may be one of the mechanisms responsible for the slow adsorption of As(v) on H/Fe. At the same time, the As(v) removal effect of H/Fe is greatly restricted by the pH. Electrostatic adsorption, adsorption co-precipitation and redox reactions act together on H/Fe in the As(v) removal process. This study provides a basis for further clarifying the adsorption, adsorption rules and mechanism of As(v) on H/Fe and a feasible method for the improvement of As(v) removal efficiency of zero-valent iron materials.

The environmental and health impacts caused by arsenic (As) in wastewater make it necessary to carefully manage As wastes.     

Thermodynamic,structural and dynamic properties of ionic liquids [C4mim][CF3COO], [C4mim][Br] in the condensed phase,using molecular simulations

In this work a series of thermodynamic, structural, and dynamical properties for the 1-butyl-3-methylimidazolium trifluoroacetate ([C₄mim][CF₃COO]) and 1-butyl-3-methylimidazolium bromide, ([C₄mim][Br]) ionic liquids (ILs) were calculated using Non-polarizable Force Fields (FF), parameterized using a methodology developed previously within the research group, for condensed phase applications. Properties such as the Vapor-Liquid Equilibrium (VLE) curve, critical points (ρ_c, T_c), Radial, Spatial and Combined Distribution Functions and self-diffusion coefficients were calculated using Equilibrium Molecular Dynamics simulations (EMD); other properties such as shear viscosities and thermal conductivities were calculated using Non-Equilibrium Molecular Dynamics simulations (NEMD). The results obtained in this work indicated that the calculated critical points are comparable with those available in the literature. The calculated structural information for these two ILs indicated that the anions interact mainly with hydrogen atoms from both the imidazolium ring and the methyl chain; the bromide anion displays twice the hydrogen coordination number than the oxygen atoms from the trifluoroacetate anion. Furthermore, Non-Covalent interactions (NCI index), determined by DFT calculations, revealed that some hydrogen bonds in the [C₄mim][Br] IL displayed similar strength to those in the [C₄mim][CF₃COO] IL, in spite of the shorter O⁻–H distances found in the latter IL. The majority of the calculated transport properties presented reasonable agreement with the experimental available data. Nonetheless, the self-diffusion coefficients determined in this work are under-estimated with respect to experimental values; however, by escalating the electrostatic atomic charges for the anion and cation to ±0.8e, only for this property, a remarkable improvement was obtained. Experimental evidence was recovered for most of the calculated properties and to the best of our knowledge, some new predictions were done mainly in thermodynamic states where data are not available. To validate the FF, developed previously within the research group, dynamic properties were also evaluated for a series of ILs such as [C₄mim][PF₆], [C₄mim][BF₄], [C₄mim][OMs], and [C₄mim][NTf₂] ILs.

Non-covalent interactions, coordination numbers, RDFs, SDFs, CDFs, and transport properties for the [C₄mim][Br] and [C₄mim][CF₃COO] ionic liquids were determined.     

Cation folding and the thermal stability limit of the ionic liquid [BMIM+][BF4−] under total vacuum

Molecular dynamics simulations reveal the behavior of the bimodal distribution of cation conformations (folded/unfolded) in ionic liquids based on alkylated imidazoles, such as [BMIM⁺][BF₄⁻]. The alkyl chains of the cations can fold and block interactions between the cations and anions, thereby reducing the cohesivity of the liquid. At room temperature, the folded conformations represent less than one-third of the total conformations. In contrast to the behavior observed during the thermal denaturation of proteins, in ionic liquids, the concentration of folded cations grows when the temperature increases. At the equimolar concentration, the system reaches the reported experimental temperature of thermal stability (similar to the thermal denaturation behavior). There is an outermost layer of cations at the interface that can tilt toward the interface and cover a layer of anions adsorbed at the interface. This interfacial conformation makes the system stable in transverse directions and unstable in the normal direction at temperatures in the region of thermal instability, limiting the rate of vaporization of neutral ion pairs, which are observed as rare events at temperatures as low as 773.15 K.

Snapshot of a vaporized neutral ion pair near the liquid layer of [BMIM⁺][BF₄⁻] under vacuum–liquid equilibrium at 773.15 K.     

Simultaneous delivery of doxorubicin and immunostimulatory CpG motif to tumors using a plasmid DNA/doxorubicin complex in mice

To achieve delivery of doxorubicin (DXR), a very commonly used anticancer agent, to tumor tissues, it was intercalated to plasmid DNA to obtain a plasmid DNA/DXR complex. The cytotoxic effects of DXR, DNA and their complex were examined in colon26/Luc cells, a murine adenocarcinoma clone stably expressing firefly luciferase, co-cultured with RAW264.7 murine macrophage-like cells. Both CpG motif-containing plasmid DNA (CpG plasmid DNA) and DXR significantly inhibited the proliferation of colon26/Luc cells, but their complex was the most effective among those examined. Non-CpG plasmid DNA was less effective than the CpG plasmid DNA. When injected into mice bearing hepatic metastases of colon26/Luc cells, the CpG plasmid DNA/DXR complex produced a significant level of IL-12 in the serum and liver. The amount of DXR delivered to tumor tissues in the liver was greater when DXR was injected as a CpG plasmid DNA/DXR complex than as free DXR. The CpG plasmid DNA/DXR complex effectively inhibited the proliferation of colon26/Luc cells in the liver compared with free DXR, CpG plasmid DNA, or non-CpG plasmid DNA/DXR complex. These results indicate that CpG plasmid DNA is an effective polymer that inhibits tumor growth by delivering both a proinflammatory signal and anticancer agent to tumor tissues.     

Green synthesis of coumarin derivatives using Brønsted acidic pyridinium based ionic liquid [MBSPy][HSO4] to control an opportunistic human and a devastating plant pathogenic fungus Macrophomina phaseolina

An eco-friendly simple protocol has been devised for the preparation of coumarin derivatives using doubly Brønsted acidic task specific ionic liquid (TSIL) as a catalyst. Solvent-free conditions were employed for the reaction of different substituted phenols with β-ketoester in TSIL to produce corresponding substituted coumarin derivatives in good to excellent yields at ambient conditions; at room temperature and with reduced reaction times. The ionic liquid catalyst can be recycled and reused up to five times. All the synthesized coumarins were evaluated for their antifungal activities against Macrophomina phaseolina, a plant as well as an opportunistic human pathogenic fungus affecting more than 500 plant species worldwide and with no registered commercial fungicide available against it, to date. Amongst all the coumarins tested, compounds 3f and 3i showed excellent antifungal activity comparable to reference fungicide mancozeb. The current methodology provides an easy and expedient way to access the coumarin core in search of potential fungicides for sustainable agriculture.

An eco-friendly simple protocol has been devised for the preparation of coumarin derivatives using doubly Brønsted acidic task specific ionic liquid (TSIL) as a catalyst.     

Physical properties and in vitro transfection efficiency of gene delivery vectors based on complexes of DNA with synthetic polycations.

Biophysical properties of polycation/DNA complexes designed for gene delivery were studied with respect to the conditions of their preparation, chemical structure and molecular weight of the polycations involved. The polycations used included a variety of cationic polymers and copolymers containing primary and tertiary amino or quaternary ammonium groups. It was found that the molecular weight and the size of these polyelectrolyte complexes (PECs) increase with increasing temperature and pH of the buffer. By decreasing the molecular weight of polycations used for PEC formation, the complexes become unstable towards coagulation in aqueous solution at lower pH. The self-assembly of DNA with low-molecular-weight polycations in water provides PECs with the lowest molecular weight, smallest size and the lowest density but their stability in NaCl solutions is very poor. Despite the complexity of the multistep transfection process, a direct correlation between the transfection efficiency in vitro and the stability of the complexes in NaCl solutions and coagulation in 0.15 M NaCl solution was found. DNA complexes with polycations containing primary amino groups showed the best stability in saline solutions and also the best transfection activity. PECs formed by polycations with quaternary ammonium groups were the least resistant to destruction by the added salt and provided the lowest activity in transfection assays. The highest transfection activity was found for DNA complexes formed with a statistical copolymer containing primary and tertiary amines.     

Mechanistic understanding of humin formation in the conversion of glucose and fructose to 5-hydroxymethylfurfural in [BMIM]Cl ionic liquid

Humin formation is one of the key issues that hinders economical 5-HMF production from hexose sugars such as glucose and fructose. In this work, the mechanism of humin formation in glucose/fructose conversion to HMF was studied in an ionic liquid system (1-butyl-3-methylimidazolium chloride, [BMIM]Cl) with CrCl₃ as the catalyst. Elemental analysis, XRD, FT-IR, and TEM were applied to study the molecular structure and morphology of the solid humins. The possible intermediates to form solid humins were investigated by HPLC-MS. We synthesized furanic model compounds that mimic the experimentally identified humin intermediates to investigate the mechanism of humin growth at an early stage. The results showed that a furan compound bearing a hydroxymethyl and an electron-donating group was unstable due to three types of reactions: (1) bimolecular ether formation reactions; (2) intermolecular addition reaction; (3) furan ring opening reaction with water. The stability of a furan compound in [BMIM]Cl was increased when the hydroxymethyl group of a furan compound was protected by a methyl group, and the stability was further enhanced with an additional electron-withdrawing group (such as an aldehyde group) on the furan ring. Protecting the hydroxymethyl group of 5-HMF with a methyl group allows easy separation of the products from the [BMIM]Cl solvent through extraction.

This study provided a new mechanistic understanding of humin formation during 5-HMF production from hexose in ionic liquids.     

Anion–cation co-operative catalysis by artificial sweetener saccharine-based ionic liquid for sustainable synthesis of 3,4-dihydropyrano[c]chromenes, 4,5-dihydropyrano[4,3-b]pyran and tetrahydrobenzo[b]pyrans in aqueous medium

In this study, a saccharine-based ionic liquid [Bmim]Sac has been found to be a sustainable catalyst for the synthesis of 3,4-dihydropyrano[c]chromenes, 4,5-dihydropyrano[4,3-b]pyran and tetrahydrobenzo[b]pyrans scaffolds through Domino Knoevenagel–Michael reaction. The easy recovery of the catalyst and high yield of the products make the protocol attractive, sustainable and economical. A mechanistic hypothesis is discussed using the concept of cooperative catalysis based on the dual (electrophilic/nucleophilic) activation of reactants by [Bmim]Sac. Furthermore, dual hydrogen bonding of saccharinate anions plays an important role in the activation of nucleophiles.

Artificial sweetener saccharine based ionic for sustainable synthesis of 3,4-dihydropyrano[c]chromenes, 4,5-dihydropyrano[4,3-b]pyran and tetrahydrobenzo[b]pyrans in aqueous medium scaffolds through Domino Knoevenagel–Michael reaction.     

Development of safe and efficient novel nonviral gene transfer using ultrasound: enhancement of transfection efficiency of naked plasmid DNA in skeletal muscle

Although clinical trials of stimulation of angiogenesis by transfection of angiogenic growth factors using naked plasmid DNA or adenoviral vector have been successful, there are still unresolved problems for human gene therapy such as low transfection efficiency and safety. From this viewpoint, it is necessary to develop safe and efficient novel nonviral gene transfer methods. As therapeutic ultrasound induces cell membrane permeabilization, ultrasound irradiation might increase the transfection efficiency of naked plasmid DNA into skeletal muscle. Thus, we examined the transfection efficiency of naked plasmid DNA using ultrasound irradiation with echo contrast microbubble (Optison) in vitro and in vivo experiments. First, we examined the feasibility of ultrasound-mediated transfection of naked plasmid DNA into skeletal muscle cells. Luciferase plasmid mixed with or without Optison was transfected into cultured human skeletal muscle cells using ultrasound (1 MHz; 0.4 W(2)) for 30 s. Interestingly, luciferase activity was markedly increased in cells treated with Optison, while little luciferase activity could be detected without Optison (P < 0.01). Electron microscopy demonstrated the transient formation of holes (less than 5 microM) in the cell surface, which could possibly explain the rapid migration of the transgene into the cells. Next, we studied the in vivo transfection efficiency of naked plasmid DNA using ultrasound with Optison into skeletal muscle. Two days after transfection, luciferase activity in skeletal muscle transfected with Optison using ultrasound was significantly increased about 10-fold as compared with plasmid alone. Successful transfection was also confirmed by beta-galactosidase staining. Finally, we examined the feasibility of therapeutic angiogenesis using naked hepatocyte growth factor (HGF) plasmid in a rabbit ischemia model using the ultrasound-Optison method. Five weeks after transfection, the angiographic score and the number of capillary density in rabbits transfected with Optison using ultrasound was significantly increased as compared with HGF plasmid alone (P < 0.01), accompanied by a significant increase in blood flow and blood pressure ratio (P < 0.01). Overall, the ultrasound transfection method with Optison enhanced the transfection efficiency of naked plasmid DNA in vivo as well as in vitro. Transfection of HGF plasmid by the ultrasound-Optison method could be useful for safe clinical gene therapy to treat peripheral arterial disease without a viral vector system.     

Evaluation of current activities of fluoroquinolones against gram-negative bacilli using centralized in vitro testing and electronic surveillance

Given the propensity for Enterobacteriaceae and clinically significant nonfermentative gram-negative bacilli to acquire antimicrobial resistance, consistent surveillance of the activities of agents commonly prescribed to treat infections arising from these organisms is imperative. This study determined the activities of two fluoroquinolones, levofloxacin and ciprofloxacin, and seven comparative agents against recent clinical isolates of Enterobacteriaceae, Pseudomonas aeruginosa, Acinetobacter baumannii, and Stenotrophomonas maltophilia using two surveillance strategies: 1) centralized in vitro susceptibility testing of isolates collected from 27 hospital laboratories across the United States and 2) analysis of data from The Surveillance Network Database-USA, an electronic surveillance network comprising more than 200 laboratories nationwide. Regardless of the surveillance method, Enterobacteriaceae, P. aeruginosa, and A. baumannii demonstrated similar rates of susceptibility to levofloxacin and ciprofloxacin. Susceptibilities to the fluoroquinolones approached or exceeded 90% for all Enterobacteriaceae except Providencia spp. (相似文献   

Comparative in vitro activity of N-formimidoyl thienamycin against gram-positive and gram-negative aerobic and anaerobic species and its beta-lactamase stability.

The in vitro activity of N-formimidoyl thienamycin was determined against 800 gram-positive and gram-negative aerobic and anaerobic bacteria and compared with the activity of cefoxitin, cefazolin, cefamandole, cefotaxime, moxalactam, ampicillin, cefoperazone, and gentamicin. N-Formimidoyl thienamycin inhibited the majority of organisms at concentrations below 1 microgram/ml. It inhibited methicillin-resistant Staphylococcus aureus, Staphylococcus epidermidis, and Streptococcus faecalis. It inhibited beta-lactamase-producing Haemophilus influenzae and Neisseria gonorrhoeae. Unlike other new beta-lactams, it inhibited Listeria. Escherichia coli, Klebsiella pneumoniae, enterobacters, Serratia, indole-positive Proteus, Acinetobacter, Pseudomonas aeruginosa, and Bacteroides resistant to other agents were inhibited. There was minimal effect of inoculum size and aerobic versus anaerobic conditions, and serum had no effect on activity. Most minimal bactericidal concentrations were two- or fourfold greater than the minimal inhibitory concentration. N-Formimidoyl thienamycin showed partial synergy with aminoglycosides against S. aureus, S. faecalis, and many Pseudomonas and Enterobacteriaceae. It was not hydrolyzed by plasmid-mediated and chromosomal beta-lactamases.     

Evaluation of 3D blood flow patterns and wall shear stress in the normal and dilated thoracic aorta using flow-sensitive 4D CMR

Background

The purpose of this study was to investigate 3D flow patterns and vessel wall parameters in patients with dilated ascending aorta, age-matched subjects, and healthy volunteers.

Methods

Thoracic time-resolved 3D phase contrast CMR with 3-directional velocity encoding was applied to 33 patients with dilated ascending aorta (diameter ≥40 mm, age=60±16 years), 15 age-matched normal controls (diameter ≤37 mm, age=68±7.5 years) and 15 young healthy volunteers (diameter ≤30 mm, age=23±2 years). 3D blood flow was visualized and flow patterns were graded regarding presence of supra-physiologic-helix and vortex flow using a semi-quantitative 3-point grading scale. Blood flow velocities, regional wall shear stress (WSS), and oscillatory shear index (OSI) were quantified.

Results

Incidence and strength of supra-physiologic-helix and vortex flow in the ascending aorta (AAo) was significantly higher in patients with dilated AAo (16/33 and 31/33, grade 0.9±1.0 and 1.5±0.6) than in controls (2/15 and 7/15, grade 0.2 ± 0.6 and 0.6 ± 0.7, P<.05) or healthy volunteers (1/15 and 0/15, grade 0.1 ± 0.3 P<.05). Greater strength of the ascending aortic helix and vortex flow were associated with significant differences in AAo diameters (P<.05). Peak systolic WSS in the ascending aorta and aortic arch was significantly lower in patients with dilated AAo (P<.0157-.0488). AAo diameter positively correlated to time to peak systolic velocities (r=0.30-0.53, P<.04), OSI (r=0.33-0.49, P<0.02) and inversely correlated to peak systolic WSS (r=0.32-0.40, P<.03). Peak systolic WSS was significantly lower in AAo aneurysms at the right and outer curvature within the AAo and proximal arch (P<.01-.05).

Conclusions

Increase in AAo diameter is significantly correlated with the presence and strength of supra-physiologic-helix and vortex formation in the AAo, as well with decrease in systolic WSS and increase in OSI.     

苯并[a]芘神经毒作用及对小鼠脑组织热应激蛋白70,90β水平的影响

背景苯并[a]芘代表的多环芳烃是一种广泛存在于生产和生活环境中的化学污染物,国外发现苯并[a]芘在一定条件下具有体外神经毒性.目的研究苯并[a]芘对小鼠的神经毒性及对脑组织中两种热应激蛋白70,90β表达的影响.设计以实验动物为研究对象的随机对照实验.单位一所大学的热生物与分子毒理实验室及一所大学的预防医学系.材料实验在华中科技大学同济医学院热生物与分子毒理实验室进行.将50只昆明种小鼠随机分为5组,10只/组,染毒组分为高、中、低3个剂量组,设溶剂对照及空白对照组.染毒组用苯并[a]芘植物油溶剂腹腔注射,高、中、低剂量组每次分别注射7.8,3.2和1.3 mg/kg,4次/周.溶剂对照组用植物油溶剂作平行处理,空白对照组不做处理.方法观察动物一般情况及神经系统表现,8周后取各组小鼠全脑组织,计算脑组织脏器系数,Western blot法检测两种热应激蛋白70,90β水平.主要观察指标苯并[a]芘染毒对小鼠脑组织热应激蛋白70及90β表达的影响.结果①中、高剂量苯并[a]芘染毒组小鼠脑组织质量明显低于空白对照组(P＜0.01,P＜0.001),高剂量BaP染毒组小鼠脑组织脏器系数明显低于其他组(P＜0.001).②热应激蛋白70表达改变以低剂量苯并[a]芘染毒组相对表达值明显增高为特征;热应激蛋白90β表达改变为中、高剂量苯并[a]芘染毒组相对表达值升高.结论苯并[a]芘具有一定的神经毒性.随着染毒剂量增加,热应激蛋白90β表达水平增高,一定条件下可作为毒理学损害标志.     

苯并[a]芘与铅单独及联合作用对体外神经元存活率及胞核DNA损伤的影响

背景苯并[a]芘对中枢神经和外周神经具有一定的损伤作用,苯并[a]芘与其他毒物联合神经毒作用的研究处于起始阶段.目的应用分子生物学技术与神经元培养相结合的手段研究苯并[a]芘、铅单独及联合作用下对体外神经细胞的毒性及胞核DNA的损伤情况.设计重复测量设计.单位重庆医科大学劳动卫生教研室和华中科技大学同济医学院职业医学研究所热生物与分子毒理实验室.材料实验于2003-06/09在华中科技大学同济医学院职业医学研究所热生物与分子毒理实验室完成,选择10只8日龄SD大鼠小脑组织制备原代细胞培养物,分10组培养,每组5个培养皿.按以下分组进行处理[1]空白对照组.[2]溶剂对照组(等量二甲基亚砜+肝微粒体酶混合物平行处理).[3]低浓度铅染毒组(醋酸铅5 μmol/L).[4]高浓度铅染毒组(醋酸铅50μmol/L).[5]低浓度苯并[a]芘染毒组(苯并[a]芘5 μmol/L+肝微粒体酶混合物).[6]高浓度苯并[a]芘染毒组(苯并[a]芘50μmol/L+肝微粒体酶混合物).[7]低浓度铅+低浓度苯并[a]芘联合染毒组.[8]低浓度铅+高浓度苯并[a]芘联合染毒组.[9]高浓度铅+低浓度苯并[a]芘联合染毒组.[10]高浓度铅+高浓度苯并[a]芘联合染毒组.方法染毒90min,胰酶消化法收集标本,椎虫蓝染色法检测各组细胞存活率;单细胞凝胶电泳法检测各组细胞胞核DNA的损伤情况.主要观察指标苯并[a]芘、铅单独及联合染毒下体外神经细胞的存活率及胞核DNA的损伤率. 结果[1]两种浓度的苯并[a]芘、铅单独或联合染毒各组细胞存活率均低于对照组[染毒组为(44.14±4.80)%～(82.40±2.70)%,对照组为(88.44±2.53)%～(90.12±2.23)%,P＜0.05～0.01].[2]高浓度苯并[a]芘单独染毒组、低浓度铅+高浓度苯并[a]芘染毒组、高浓度铅+低浓度苯并[a]芘染毒组、高浓度铅+高浓度苯并[a]芘染毒组胞核DNA损伤程度均高于对照组[63%(19/30),87%(26/30),80%(24/30),97%(29/30),13%(4/30),20%(6/30),P＜0.01].结论苯并[a]芘与铅均有一定的体外神经毒性,且二者有协同作用;苯并[a]芘损害体外培养神经元胞核DNA的能力大于铅.     

苯并[a]芘与铅单独及联合作用对体外神经元存活率及胞核DNA损伤的影响

背景：苯并[a]芘对中枢神经和外周神经具有一定的损伤作用，苯并[a]芘与其他毒物联合神经毒作用的研究处于起始阶段。目的：应用分子生物学技术与神经元培养相结合的手段研究苯并[a]芘、铅单独及联合作用下对体外神经细胞的毒性及胞核DNA的损伤情况。设计：重复测量设计。单位：重庆医科大学劳动卫生教研室和华中科技大学同济医学院职业医学研究所热生物与分子毒理实验室。材料：实验于2003-06／09在华中科技大学同济医学院职业医学研究所热生物与分子毒理实验室完成，选择10只8日龄SD大鼠小脑组织制备原代细胞培养物，分10组培养，每组5个培养皿。按以下分组进行处理：①空白对照组。②溶剂对照组（等量二甲基亚砜＋肝微粒体酶混合物平行处理）。③低浓度铅染毒组（醋酸铅5μmol／L）。④高浓度铅染毒组（醋酸铅50μmol／L）。⑤低浓度苯并[a]芘染毒组（苯并[a]芘5μmol／L＋肝微粒体酶混合物）。⑥高浓度苯并[a]芘染毒组（苯并[a]芘50μmol／L＋肝微粒体酶混合物）。⑦低浓度铅＋低浓度苯并[a]芘联合染毒组。⑧低浓度铅＋高浓度苯并[a]芘联合染毒组。⑨高浓度铅＋低浓度苯并[a]芘联合染毒组。⑩高浓度铅＋高浓度苯并[a]芘联合染毒组。方法：染毒90min，胰酶消化法收集标本，椎虫蓝染色法检测各组细胞存活率；单细胞凝胶电泳法检测各组细胞胞核DNA的损伤情况。主要观察指标：苯并[a]芘、铅单独及联合染毒下体外神经细胞的存活率及胞核DNA的损伤率。结果：①两种浓度的苯并[a]芘、铅单独或联合染毒各组细胞存活率均低于对照组[染毒组为（44．14&;#177;4．80%～（82.40＋2．70）％，对照组为（88．44&;#177;2.53）％～（90．12&;#177;2.23％，P〈0．05～0．01]。②高浓度苯并[a]芘单独染毒组、低浓度铅＋高浓度苯并[a]芘染毒组、高浓度铅＋低浓度苯并[a]芘染毒组、高浓度铅＋高浓度苯并[a]芘染毒组胞核DNA损伤程度均高于对照组[63％（19／30），87％（26／30），80％（24/30），97％（29／30），13％（4／30），20％（6／30），P〈0．01]。结论：苯并[a]芘与铅均有一定的体外神经毒性，且二者有协同作用；苯并[a]芘损害体外培养神经元胞核DNA的能力大于铅。     

In vivo quantification of dopamine transporters in mice with unilateral 6-OHDA lesions using [11C]methylphenidate and PET

Quantification of the binding of [¹¹C]methylphenidate to the dopamine transporter (DAT) using positron emission tomography (PET) is often used to evaluate the integrity of dopaminergic neurons in the striatal regions of the brain. Over the past decade, many genetically engineered mouse models of human disease have been developed and have become particularly useful for the study of disease onset and progression over time. Quantitative imaging of small structures such as the mouse brain is especially challenging. Thus, the aims of this study were (1) to evaluate the accuracy of quantifying DAT binding using in vivo PET and (2) to examine the impact of different methodologies.

Methods

Eight mice were scanned with [¹¹C]methylphenidate under true or transient equilibrium conditions using a bolus and constant infusion protocol or a bolus injection protocol to evaluate the accuracy of the Logan graphical approach for [¹¹C]methylphenidate imaging in mice. Displacement with unlabeled methylphenidate (0.1, 3 and 10 mg/kg) was used to verify specific binding. In a second experiment, 30 mice were lesioned by injection of 6-hydroxydopamine (6-OHDA) at doses of 0, 2 or 4 μg (n = 10) into the right striatum to assess the dose-dependent correlation between the PET signal and dopaminergic degeneration. In addition, we performed test-retest experiments and used ex vivo autoradiography (AR) to validate the effect of partial volume on the accuracy of the [¹¹C]methylphenidate PET quantification in the mouse striatum.

Results

The binding potentials (BP_ND) calculated from the Logan graphical analysis under transient equilibrium conditions (1.03 ± 0.1) were in excellent agreement with those calculated at true equilibrium (1.07 ± 0.1). Displacement of specific binding with 0.1, 3 and 10 mg/kg methylphenidate resulted in 38%, 77% and 81% transporter occupancy in the striatum. Intra-striatal injections of 6-OHDA caused a dose-dependent decrease in the specific binding of [¹¹C]methylphenidate to the DAT in the striatum. The BP_ND was reduced by 49% and 61% after injection with 2 and 4 μg of 6-OHDA, respectively. The test-retest reproducibility was 6% in the healthy striatum and 27% in the lesioned striatum. In addition, only a small (15%) difference was found between the [¹¹C]methylphenidate DVR-1 values determined by PET and AR on the healthy side, and no differences were observed on the lesioned side.

Conclusion

The present work demonstrates for the first time that [¹¹C]methylphenidate PET is useful for the quantification of striatal dopamine transporters at the dopaminergic nerve terminals in the mouse striatum; therefore, this marker may be used as a biomarker in genetically engineered mouse models of neurodegenerative disorders. However, only changes resulting in greater than 10% differences in BP_ND values can reliably be detected in vivo.     

Pronounced in vitro and in vivo antiretroviral activity of 5-substituted 2,4-diamino-6-[2-(phosphonomethoxy)ethoxy] pyrimidines

OBJECTIVES: To discover new potent and selective anti-human immunodeficiency virus (HIV) acyclic nucleoside phosphonate (ANP) drugs with in vivo antiretroviral activity. METHODS: New acyclic pyrimidine nucleoside phosphonate derivatives that mimic the structure of the anti-HIV purine nucleoside phosphonates 9-(2-phosphonylmethoxyethyl)adenine (PMEA, adefovir) and (R)-9-(2-phosphonylmethoxypropyl)adenine (PMPA, tenofovir) were designed by linking the acyclic side chain of the ANPs through an ether bond to the C-6 position instead of the N-1 position of the pyrimidine ring. The compounds were evaluated against HIV and Moloney murine sarcoma virus (MSV) in cell culture, including a broad variety of HIV-1 clade clinical isolates and relevant mutant (drug-resistant) HIV-1 isolates. Their antiviral activities were correlated and investigated in an in vivo model consisting of MSV-infected newborn mice. MSV-induced tumour formation and associated death were recorded in drug-treated animals. RESULTS: Several 5-substituted 6-[2-(phosphonomethoxy)ethoxy]-2,4-diaminopyrimidine (PMEO-DAPy) analogues were found to inhibit a broad variety of HIV-1 clinical isolates. They showed a more favourable cross-resistance profile to mutant virus isolates than adefovir and tenofovir. There was a close correlation between inhibition of MSV in C3H/3T3 cells and inhibition of HIV-1 in CEM cells. The PMEO-DAPy derivatives potently inhibited MSV-induced tumour cell formation in newborn mice. The 5-methyl analogue PMEO-5-Me-DAPy proved markedly more inhibitory to MSV-induced tumour cell formation and associated animal death than its unsubstituted parent PMEO-DAPy derivative. When compared with adefovir, PMEO-5-Me-DAPy was less toxic and more antivirally active in MSV-infected mice. CONCLUSIONS: PMEO-5-Me-DAPy deserves further (pre)clinical investigations as a candidate anti-HIV drug.