Forced Oxidative Degradation Pathways of the Imidazole Moiety of Daclatasvir

Daclatasvir hydrochloride (DCV) is the active pharmaceutical ingredient of Daklinza, a marketed product for the treatment of hepatitis C viral infection. The intrinsic stability of daclatasvir was evaluated via a forced degradation study. DCV was found to be stable in the solid state. In solution, its carbamate moiety is susceptible to basic hydrolysis, whereas its imidazole is liable to base-mediated autoxidation to form degradants 1 and 3, 7-8, respectively. The imidazole moiety can also be oxidized to form degradants 6-7 in the presence of hydrogen peroxide or azobisisobutyronitrile. The chloro-adduct degradant 9 was also observed in hydrogen peroxide solution. Furthermore, the imidazole moiety is sensitive to photodegradation in solution. Degradants 2-8 were observed in a solution of DCV exposed to high intensity light/UV light; the formation of degradants 2 and 5-8 was postulated through 4 degradation pathways. The degradants 3 and 4 were deemed to be secondary degradants of 7 and 5, respectively.     

Marmoset cytochrome P450 2B6, a propofol hydroxylase expressed in liver

1. The common marmoset (Callithrix jacchus) is a useful experimental animal to evaluate the pharmacokinetics of drug candidates. Cytochrome P450 (P450) 2B enzyme in marmoset livers has been identified; however, only limited information on the enzymatic properties and distribution has been available.

2. Marmoset P450 2B6 amino acids showed high sequence identities (>86%) with those of primates including humans and cynomolgus monkeys. Phylogenetic analysis using amino acid sequences indicated that marmoset P450 2B6 was closer to human and cynomolgus monkey P450 2B6 than to P450 2B orthologs of other species, including pigs, dogs, rabbits and rodents.

3. Quantitative polymerase chain reaction analysis using specific primers showed P450 2B6 mRNA predominantly expressed in livers among the five marmoset tissues, similar to those of humans and cynomolgus monkeys.

4. Marmoset P450 2B6 heterologously expressed in Escherichia coli membranes oxidized 7-ethoxycoumarin, pentoxyresorufin, propofol and testosterone, at roughly similar rates to those of humans and/or cynomolgus monkeys. A high capacity of marmoset P450 2B6 with propofol 4-hydroxylation (at low ionic strength conditions) with a low K_m value was relatively comparable to that for marmoset livers.

5. These results collectively indicated a high propofol 4-hydroxylation activity of P450 2B6 expressed in marmoset livers.

     

Interleukin-1β and tumor necrosis factor-α affect cytochrome P450 expression in cynomolgus macaque hepatocytes

The cynomolgus macaque, partly due to its evolutionary closeness to humans, is an important nonhuman primate species used in drug metabolism studies. In humans, expressions of cytochromes P450 (P450s), including the important drug-metabolizing enzyme P450 3A4, are affected by various cytokines. However, this phenomenon has not been fully investigated in cynomolgus macaques. In this study, the effects of cytokines on P450 expression were investigated using the quantitative polymerase chain reaction to evaluate mRNA expression. Hepatocytes from cynomolgus macaques were treated with lipopolysaccharide and various cytokines, including interleukin (IL)-1β, IL-2, IL-6, interferon-γ, and tumor necrosis factor-α, and the expression levels of 11 P450s were compared with those of solvent-treated controls. Tumor necrosis factor-α significantly decreased cynomolgus P450 2C8 and 2C76 mRNA expression in multiple lots of cynomolgus hepatocytes investigated. IL-1β significantly decreased cynomolgus P450 1A1, 2C8, 2C19, and 2C76 mRNA expression, but increased P450 3A5 mRNA expression in multiple lots of hepatocytes. Moreover, P450 1A1-and 2C19-mediated drug oxidations were significantly and dose-dependently suppressed by IL-1β, under the present limited conditions. These results suggest that cytokines can influence hepatic P450 mRNA expression levels in cynomolgus macaques, just as cytokines are reported to affect P450 expression in humans.     

微弧氧化技术与钛合金表面成骨细胞增殖及骨向分化能力

文题释义：微弧氧化技术：是金属材料表面处理的一种技术，将金属材料(如，镁、铝、钛等)置于特定电解液中，通过弧光放电过程中瞬时的高温和高压条件产生结合于金属表面的陶瓷膜层，该种方法制备的涂层材料具有高硬度、耐磨的特点。  种植体：是针对于人体自身牙齿缺失而植入上下颌骨内的人工牙根。性能优良的种植体需要同时具备高强度、耐降解、生物相容性好的特性。目前复合材料类如金属表面添加陶瓷涂层材料，具有上述多种材料特性而被临床选择。 背景：采用普通电化学法可在钛及其合金表面制备纳米级羟基磷灰石涂层，但该涂层吸收降解缓慢，需要8-12周的时间。而微弧氧化可在复杂表面形成均匀薄膜，有利于细胞黏附和骨组织长入。 目的：探索微弧氧化羟基磷灰石涂层钛合金对成骨细胞增殖及骨向分化能力的影响。 方法：采用电化学法与微弧氧化法分别制备羟基磷灰石涂层钛合金材料，检测两种材料表面的接触角。将成骨细胞系hFOB1.19接种于两组羟基磷灰石涂层钛合金材料表面，培养48 h时，采用扫描电镜观察细胞在材料上的形态变化；培养1，12，24，48，72 h时，采用MTT法检测细胞增殖；培养1，3，5 d时，比较两组材料表面细胞计数与碱性磷酸活性；培养第5天，采用Western Blotting检测细胞内骨形态发生蛋白2和骨形态发生蛋白4表达。 结果与结论：①微弧氧化组材料表面的接触角小于电化学组[(66.5±2.2)°，(52.8±2.1)°，P=0.001 5)]；②扫描电镜显示，电化学组成骨细胞形态不规则，胞体皱缩不饱满，与材料贴合较差；微弧氧化组细胞充分伸展，形态扁平，与材料贴合紧密；③微弧氧化组12-72 h的细胞增殖快于电化学组(P < 0.05)，培养3，5 d的细胞计数多于电化学组(P < 0.05)；④微弧氧化组培养1，3，5 d的细胞碱性磷酸酶活性高于电化学组(P < 0.05)；⑤微弧氧化组骨形态发生蛋白2和骨形态发生蛋白4表达均高于电化学组(P < 0.05)；⑥结果表明，微弧氧化羟基磷灰石涂层钛合金材料可促进成骨细胞的增殖及骨向分化能力。 ORCID: 0000-0003-2787-4667(王艳玲) 中国组织工程研究杂志出版内容重点：生物材料；骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性；组织工程     

Coupling of diversification and pH adaptation during the evolution of terrestrial Thaumarchaeota

The Thaumarchaeota is an abundant and ubiquitous phylum of archaea that plays a major role in the global nitrogen cycle. Previous analyses of the ammonia monooxygenase gene amoA suggest that pH is an important driver of niche specialization in these organisms. Although the ecological distribution and ecophysiology of extant Thaumarchaeota have been studied extensively, the evolutionary rise of these prokaryotes to ecological dominance in many habitats remains poorly understood. To characterize processes leading to their diversification, we investigated coevolutionary relationships between amoA, a conserved marker gene for Thaumarchaeota, and soil characteristics, by using deep sequencing and comprehensive environmental data in Bayesian comparative phylogenetics. These analyses reveal a large and rapid increase in diversification rates during early thaumarchaeotal evolution; this finding was verified by independent analyses of 16S rRNA. Our findings suggest that the entire Thaumarchaeota diversification regime was strikingly coupled to pH adaptation but less clearly correlated with several other tested environmental factors. Interestingly, the early radiation event coincided with a period of pH adaptation that enabled the terrestrial Thaumarchaeota ancestor to initially move from neutral to more acidic and alkaline conditions. In contrast to classic evolutionary models, whereby niches become rapidly filled after adaptive radiation, global diversification rates have remained stably high in Thaumarchaeota during the past 400–700 million years, suggesting an ongoing high rate of niche formation or switching for these microbes. Our study highlights the enduring importance of environmental adaptation during thaumarchaeotal evolution and, to our knowledge, is the first to link evolutionary diversification to environmental adaptation in a prokaryotic phylum.Ammonia oxidation, the first and rate-limiting step in nitrification, is central to the global nitrogen cycle. Ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB) perform this biochemical transformation, converting as much as 70% of the 100 Tg of nitrogen fertilizer applied annually (1) and generating significant nitrous oxide (2). Although only distantly related, AOA and AOB perform similar ecosystem functions by catalyzing the conversion of ammonia to nitrite (via hydroxylamine) by using ammonia monooxygenase, a multimeric enzyme comprising the subunits AmoA, AmoB, and AmoC (3). Although AOA were initially classified as Crenarchaeota, improvements in phylogenetic methods and genomic sampling of uncultured archaeal diversity led to their placement within a new archaeal phylum, the Thaumarchaeota (4, 5). These organisms are ubiquitous, with archaeal amoA genes frequently outnumbering those of AOB (6). Furthermore, AOA are the principal drivers of ammonia oxidation in many soils, particularly those with low pH (7, 8). Environmental pH is a major factor affecting the distribution of AOA in terrestrial ecosystems (3, 9), and most terrestrial Thaumarchaeota can be phylogenetically assigned to one of five pH-adapted lineages, two being acidophilic, two alkalinophilic, and one neutrophilic (9).Despite great progress in understanding of thaumarchaeotal ecology and physiology (10), little is known about the evolutionary mechanisms that have generated their great diversity in nature. In multicellular eukaryotes, the fossil record can provide insight into evolutionary processes over geological timescales, but such approaches are not possible for many prokaryotes because of the lack of an informative fossil record. In this context, recently developed probabilistic methods geared toward reconstructing the dynamics of species diversification and trait evolution using molecular phylogenies (11–13) have great potential. A recent Bayesian method that explicitly aims to characterize and quantify heterogeneity in evolutionary rates (13–15) holds particular promise for modeling prokaryotic evolution, whereby features such as massive population size, frequent lateral gene transfer (LGT), and fast growth all facilitate rapid increases in diversity, suggesting that complex evolutionary regimes may be the norm. Here, we apply these recently developed methods to test a key hypothesis about the ecological drivers of thaumarchaeotal diversification through deep evolutionary time. Based on the documented importance of soil pH in structuring modern AOA communities (9), we tested the hypothesis that adaptation to pH is an important driver of diversification in the Thaumarchaeota by comparing associations between pH and a range of other environmental factors with diversification in Thaumarchaeota.     

Attachment of gold nanoparticles to glassy carbon electrode and its application for the voltammetric resolution of ascorbic acid and dopamine

Gold nanoparticles have been attached on glassy carbon electrode surface through sulfhydryl-terminated monolayer and the gold nanoparticles-immobilized glassy carbon electrodes have been applied to the electrocatalytic oxidation of ascorbic acid, reducing the overpotential by about 200 mV with obviously increased current response. Due to its strong electrocatalytic activity towards ascorbic acid, the gold nanoparticles modified electrode can resolve the overlapped voltammetric waves of ascorbic acid and dopamine into two well-defined voltammetric peaks with peak-to-peak separation in potentials of about 300 mV. This can be used to allow the selective determination of ascorbic acid in the presence of dopamine. The catalytic current obtained from differential pulse voltammetry is linearly dependent on ascorbic acid concentration over the range of 6.5 × 10^?6 to 1.45 × 10^?4 M with correlation coefficient of 0.998 in the presence of dopamine. The detection limit (3σ) for AA was found to be 2.8 × 10^?6 M. The simultaneous determination of ascorbic acid and dopamine in their binary mixture has also been investigated. The modified electrode shows good selectivity, stability and anti-fouling properties. The proposed methods have been used for the selective determination of ascorbic acid in the presence of dopamine and for the simultaneous determination of both them in their mixtures with satisfactory results.     

Electrochemical properties of a tetrabromo-p-benzoquinone modified carbon paste electrode. Application to the simultaneous determination of ascorbic acid,dopamine and uric acid

The electrochemical study of a tetrabromo-p-benzoquinone modified carbon paste electrode (TBQ-MCPE), as well as its efficiency for electrocatalytic oxidation of ascorbic acid, dopamine and uric acid, is described. Cyclic voltammetry was used to investigate the redox properties of this modified electrode at various solution pH values and at various scan rates. Three linear segments were found with slope values of ?58.4 mV/pH, ?28.1 mV/pH and 0.0 mV/pH in the pH range 2.0–7.1, pH 7.1–9.0 and pH 9.0–11.0, respectively. The apparent charge transfer rate constant, k_s, and transfer coefficient, α, for electron transfer between TBQ and CPE were calculated as 3.79 ± 0.10 s^?1 and 0.55, respectively. The electrode was also employed to study the electrocatalytic oxidation of AA, using cyclic voltammetry, chronoamperometry and differential pulse voltammetry as diagnostic techniques. It has been found that the oxidation of AA at the surface of TBQ-MCPE occurs at a potential of about 430 mV less positive than that of an unmodified CPE. The diffusion coefficient of AA was also estimated using chronoamperometry. The kinetic parameters such as the electron transfer coefficient, α, and heterogeneous rate constant, $k_{\mathrm{h}}^{\prime }$, for oxidation of AA at the TBQ-MCPE surface was determined using cyclic voltammetry. Differential pulse voltammetry (DPV) exhibits two linear dynamic ranges and a detection limit of 0.62 μM for AA. In DPV, the TBQ-MCPE could separate the oxidation peak potentials of AA, DA and UA present in the same solution, though at the unmodified CPE the peak potentials were indistinguishable. This modified electrode was quite effective not only to detect AA, DA and UA, but also in simultaneous determination of each component concentration in the mixture.     

阳极氧化处理后的新型钛合金表面成骨细胞OPG、RANKL基因表达研究

目的:新型钛合金Ti-24Nb-4Zr-7.9Sn(TNZS)经过阳极氧化(anodic oxidation,AD)技术处理后,分析其表面的人成骨样MG63细胞骨保护素(osteoprotegerin,OPG)、细胞核因子-κB受体活化因子配体(RANKL)基因表达水平.方法:将人成骨样MG63细胞接种于Ti-6Al-4V、TNZS、AD-TNZS表面,采用半定量RT-PCR法检测OPG、RANKL mRNA的表达量.结果:人成骨样MG63细胞在AD-TNZS表面的OPGm RNA表达量有所提高,而RANKL mRNA的表达量3组材料间无明显差异.结论:阳极氧化处理的TNZS钛合金可能通过影响骨保护素、细胞核因子-κB受体活化因子配体调节成骨细胞、破骨细胞的平衡,从而促进种植体植入后的骨重建.