Development of an Efficient G‐Quadruplex‐Stabilised Thrombin‐Binding Aptamer Containing a Three‐Carbon Spacer Molecule

The thrombin‐binding aptamer (TBA), which shows anticoagulant properties, is one of the most studied G‐quadruplex‐forming aptamers. In this study, we investigated the impact of different chemical modifications such as a three‐carbon spacer (spacer‐C₃), unlocked nucleic acid (UNA) and 3′‐amino‐modified UNA (amino‐UNA) on the structural dynamics and stability of TBA. All three modifications were incorporated at three different loop positions (T3, T7, T12) of the TBA G‐quadruplex structure to result in a series of TBA variants and their stability was studied by thermal denaturation; folding was studied by circular dichroism spectroscopy and thrombin clotting time. The results showed that spacer‐C₃ introduction at the T7 loop position (TBA‐SP7) significantly improved stability and thrombin clotting time while maintaining a similar binding affinity as TBA to thrombin. Detailed molecular modelling experiments provided novel insights into the experimental observations, further supporting the efficacy of TBA‐SP7. The results of this study could provide valuable information for future designs of TBA analogues with superior thrombin inhibition properties.     

Anticoagulant Oligonucleotide–Peptide Conjugates: Identification of Thrombin Aptamer Conjugates with Improved Characteristics

Oligonucleotide–peptide conjugates (OPCs) are a promising class of biologically active compounds with proven potential for improving nucleic acid therapeutics. OPCs are commonly recognized as an efficient instrument to enhance the cellular delivery of therapeutic nucleic acids. In addition to this application field, OPCs have an as yet unexplored potential for the post-SELEX optimization of DNA aptamers. In this paper, we report the preparation of designer thrombin aptamer OPCs with peptide side chains anchored to a particular thymidine residue of the aptamer. The current conjugation strategy utilizes unmodified short peptides and support-bound protected oligonucleotides with activated carboxyl functionality at the T3 thymine nucleobase. The respective modification of the oligonucleotide strand was implemented using N3-derivatized thymidine phosphoramidite. Aptamer OPCs retained the G-quadruplex architecture of the parent DNA structure and showed minor to moderate stabilization. In a series of five OPCs, conjugates bearing T3–Ser–Phe–Asn (SFN) or T3–Tyr–Trp–Asn (YWN) side chains exhibited considerably improved anticoagulant characteristics. Molecular dynamics studies of the aptamer OPC complexes with thrombin revealed the roles of the amino acid nature and sequence in the peptide subunit in modulating the anticoagulant activity.     

The Functional Role of Loops and Flanking Sequences of G-Quadruplex Aptamer to the Hemagglutinin of Influenza a Virus

Nucleic acid aptamers are generally accepted as promising elements for the specific and high-affinity binding of various biomolecules. It has been shown for a number of aptamers that the complexes with several related proteins may possess a similar affinity. An outstanding example is the G-quadruplex DNA aptamer RHA0385, which binds to the hemagglutinins of various influenza A virus strains. These hemagglutinins have homologous tertiary structures but moderate-to-low amino acid sequence identities. Here, the experiment was inverted, targeting the same protein using a set of related, parallel G-quadruplexes. The 5′- and 3′-flanking sequences of RHA0385 were truncated to yield parallel G-quadruplex with three propeller loops that were 7, 1, and 1 nucleotides in length. Next, a set of minimal, parallel G-quadruplexes with three single-nucleotide loops was tested. These G-quadruplexes were characterized both structurally and functionally. All parallel G-quadruplexes had affinities for both recombinant hemagglutinin and influenza virions. In summary, the parallel G-quadruplex represents a minimal core structure with functional activity that binds influenza A hemagglutinin. The flanking sequences and loops represent additional features that can be used to modulate the affinity. Thus, the RHA0385–hemagglutinin complex serves as an excellent example of the hypothesis of a core structure that is decorated with additional recognizing elements capable of improving the binding properties of the aptamer.     

5‐Hydroxymethyl‐2′‐Deoxyuridine Residues in the Thrombin Binding Aptamer: Investigating Anticoagulant Activity by Making a Tiny Chemical Modification

We report an investigation into analogues of the thrombin binding aptamer (TBA). Individual thymidines were replaced by the unusual residue 5‐hydroxymethyl‐2′‐deoxyuridine (hmU). This differs from the canonical thymidine by a hydroxyl group on the 5‐methyl group. NMR and CD data clearly indicate that all TBA derivatives retain the ability to fold into the “chair‐like” quadruplex structure. The presence of the hmU residue does not significantly affect the thermal stability of the modified aptamers compared to the parent, except for analogue H9 , which showed a marked increase in melting temperature. Although all TBA analogues showed decreased affinities to thrombin, H3 , H7 , and H9 proved to have improved anticoagulant activities. Our data open up the possibility to enhance TBA biological properties, simply by introducing small chemical modifications.     

Thrombin-Binding Aptamer with Inversion of Polarity Sites (IPS): Effect on DNAzyme Activity and Anticoagulant Properties

In this work we examined the properties of thrombin-binding aptamer (TBA) modified by the introduction of inversion of polarity sites (IPS) in order to assess the effect of modification on the activation of TBA to serve as DNAzyme with peroxidase-like activity. Two oligonucleotides were designed to possess one (IPS1) or three (IPS2) inversion sites. TBA typically forms antiparallel G-quadruplexes with two G-tetrads, which exhibits very low DNAzyme peroxidise activity. DNAzyme activity is generally attributed to parallel G-quadruplexes. Hence, inversion of polarity was introduced in the TBA molecule to force the change of G-quadruplex topology. All oligonucleotides were characterized using circular dichroism and UV-Vis melting profiles. Next, the activity of the DNAzymes formed by studied oligonucleotides and hemin was investigated. The enhancement of peroxidase activity was observed when inversion of polarity was introduced. DNAzyme based on IPS2 showed the highest peroxidase activity in the presence of K⁺ or NH₄⁺ ions. This proves that inversion of polarity can be used to convert a low-activity DNAzyme into a DNAzyme with high activity. Since TBA is known for its anticoagulant properties, the relevant experiments with IPS1 and IPS2 oligonucleotides were performed. Both IPS1 and IPS2 retain some anticoagulant activity in comparison to TBA in the reaction with fibrinogen. Additionally, the introduction of inversion of polarity makes these oligonucleotides more resistant to nucleases.     

Relative Nuclease Resistance of a DNA Aptamer Covalently Conjugated to a Target Protein

A major obstacle to the therapeutic application of an aptamer is its susceptibility to nuclease digestion. Here, we confirmed the acquisition of relative nuclease resistance of a DNA-type thrombin binding aptamer with a warhead (TBA₃) by covalent binding to a target protein in the presence of serum/various nucleases. When the thrombin-inhibitory activity of TBA₃ on thrombin was reversed by the addition of the complementary strand, the aptamer was instantly degraded by the nucleases, showing that the properly folded/bound aptamer conferred the resistance. Covalently binding aptamers possessing both a prolonged drug effect and relative nuclease resistance would be beneficial for in vivo translational applications.     

卡介菌多糖核酸治疗过敏性鼻炎合并哮喘的临床疗效

目的观察卡介菌多糖核酸对过敏性鼻炎合并哮喘的临床治疗效果。方法将98例2001年初～2009年初在吉林市中心医院耳鼻喉科就诊的过敏性鼻炎合并哮喘患者随机分为治疗1组、治疗2组及对照组,3组均给予相同的过敏哮喘基础治疗,治疗1组再经肌肉注射卡介菌多糖核酸(BCG polysaccharide and nucleic acid,BCG-PSN)注射液,疗程3个月;治疗2组再经肌肉注射BCG-PSN,疗程6个月;对照组只进行基础治疗,不给予BCG-PSN。观察疗程结束后1年内3组患者过敏性鼻炎及哮喘的发作情况,根据哮喘的发作次数及程度判断疗效。结果疗程结束后0～6个月内,治疗1组和治疗2组的过敏性鼻炎及哮喘的临床症状明显好转,显效率和总有效率均明显高于对照组(P<0.05或P<0.01);疗程结束后7～12个月,治疗2组的显效率和总有效率均明显高于对照组(P均<0.05)。BCG-PSN治疗中未见明显不良反应。结论BCG-PSN治疗过敏性鼻炎合并哮喘疗效确切,安全可靠,远期疗效与疗程有关。     

凤鸣村腐植酸和活性褐煤对Cr(Ⅵ)离子的吸附等温式

研究了从凤鸣村褐煤中提取的腐植酸和活性褐煤对Cr (Ⅵ )离子的吸附 ,求出了Langmuir和Fre undlich方程的有关参数 ,结果表明 ,腐植酸对Cr(Ⅵ )离子吸附具有以化学吸附为主的Langmuir单分子吸附特征 ,活性褐煤对Cr(Ⅵ )离子吸附符合Freundlich方程。     

Influence of a Diet Enriched with Perilla Seed Bran on the Composition of Omega-3 Fatty Acid in Nile Tilapia

This study evaluated the effect of inclusion of perilla seed bran (PSB) in the diet of Nile tilapia genetically improved farmed tilapia (GIFT) on the concentration of fatty acid n‐3 polyunsaturated fatty acids (PUFAs) according to the function of feeding time. The GIFT were cultivated in net cages for 60 days using a control diet with soybean oil and supplemented with PSB. Analyses of the proximate composition and quantification of fatty acids (mg g^?1 of total lipids) were performed in muscle tissue every 15 days. The PSB diet influenced the lipid composition of GIFT fillets by linolenic acid incorporation, which was approximately 384 %, resulting in an increase of 5.2 times the sum of n‐3 PUFA. On the other hand, there was a decrease in the sum of saturated fatty acids. During treatment, there was a continuous increase in n‐3 PUFA, proving the influence of feeding time in the lipid composition of GIFT fillets. The indices of the lipid quality of fillets coming from fish fed the PSB diet were improved. Of these indices, a n‐6/n‐3 ratio presented a significant reduction of 74.15 %, proving the quality of the dietary lipid. Therefore, the inclusion of PSB significantly altered the fatty acid muscle tissue composition of GIFT during feeding time, contributing to an increase in its nutritional value.     

MNT废酸用甲苯萃取工艺的探讨

介绍硝基甲苯废酸用甲苯萃取的工艺选择, 经萃取可回收废酸中的硝酸和硝基化合物。废酸不需脱硝即可直接进行浓缩。     

Structure-Activity Relationship Explorations and Discovery of a Potent Antagonist for the Free Fatty Acid Receptor 2

Free fatty acid receptor 2 (FFA2) is a sensor for short-chain fatty acids that has been identified as an interesting potential drug target for treatment of metabolic and inflammatory diseases. Although several ligand series are known for the receptor, there is still a need for improved compounds. One of the most potent and frequently used antagonists is the amide-substituted phenylbutanoic acid known as CATPB ( 1 ). We here report the structure-activity relationship exploration of this compound, leading to the identification of homologues with increased potency. The preferred compound 37 (TUG-1958) was found, besides improved potency, to have high solubility and favorable pharmacokinetic properties.     

过氧化氢对D-氨基酸氧化酶转化头孢菌素C为戊二酰-7-氨基头孢烯酸反应的影响

在D-氨基酸氧化酶D1AAO转化头孢菌素C(CPC)为戊二酰基-7-氨基头孢烯酸(Gl-7-ACA)的反应中,所生成的过氧化氢同时与CPC和Gl-7-ACA发生的氧化副反应是造成目标产物收率损失的关键因素之一.通过联用溶氧电极和过氧化氢电极,测定了DAAO转化CPC为Gl-7-ACA的反应体系内溶氧浓度与相应的过氧化氢浓度的变化曲线,测知反应体系中过氧化氢浓度积累最高可达5 mmol·L-1.模拟该反应条件下过氧化氢的氧化反应可知,在过氧化氢浓度较低时,溶液的酸性可加速该氧化反应.在中性条件下,过氧化氢浓度为5 mmol·L-1时,反应1h后,CPC的氧化损失为2%,Gl-7-ACA的氧化损失为3%.通过改进氧气分布器、搅拌转速和并调控氧气流量以控制溶液中溶解氧的浓度,来适度地降低物系中过氧化氢积累浓度,使反应收率提高了2.2%.     

卡介菌多糖核酸注射液对继发性暂时性免疫功能低下儿童的调节作用

目的观察卡介菌多糖核酸注射液对继发性暂时性免疫功能低下儿童的调节作用。方法选择临床上反复呼吸道感染的儿童,检测T细胞亚群和免疫球蛋白,以其中至少一项指标低于同年龄段正常值最低限的52名为观察对象,给予卡介菌多糖核酸注射液治疗,疗程3个月。3个月后复查T细胞亚群和免疫球蛋白。结果治疗后细胞免疫和体液免疫均得到不同程度的改善,治疗前后各项指标相比,差异均有非常显著或显著意义。结论卡介菌多糖核酸注射液对反复呼吸道感染所致免疫功能低下儿童疗效确切。     

L-乳酸生物炼制技术的新进展

介绍乳酸炼制工艺路线的选择,即原料路线的确定依据以及国内外的发酵工艺和提取工艺路线。发酵工艺包括细菌发酵与根霉发酵、清液发酵和非清液发酵、连续发酵与间歇分批发酵以及膜耦合发酵与萃取发酵;而提取工艺也介绍了钙盐法和高真空蒸馏等5种方法。最后对南京绿天源生物化工工程有限公司的乳酸炼制工艺的流程和技术特点进行了简要介绍。     

G quadruplex-based FRET probes with the thrombin-binding aptamer (TBA) sequence designed for the efficient fluorometric detection of the potassium ion

The dual-labeled oligonucleotide derivative, FAT-0, carrying 6- carboxyfluorescein (FAM) and 6-carboxytetramethylrhodamine (TAMRA) labels at the 5' and 3' termini of the thrombin-binding aptamer (TBA) sequence 5'-GGT TGG TGT GGT TGG-3', and its derivatives, FAT-n (n=3, 5, and 7) with a spacer at the 5'-end of a TBA sequence of T(m)A (m=2, 4, and 6) have been designed and synthesized. These fluorescent probes were developed for monitoring K(+) concentrations in living organisms. Circular dichroism, UV-visible absorption, and fluorescence studies revealed that all FAT-n probes could form intramolecular tetraplex structures after binding K(+). Fluorescence resonance energy transfer and quenching results are discussed taking into account dye-dye contact interactions. The relationship between the fluorescence behavior of the probes and the spacer length in FAT-n was studied in detail and is discussed.     

A Study of the Impacts of Periodic Ozone Residuals on Biologically Active Filters

The research presented in this article investigated the effects of ozone residuals on the biological performance of a biologically active anthracite/sand filter. In addition, the impact of residuals of hydrogen peroxide (H₂O₂), added as an accelerator of natural ozone decay, was investigated. Experiments were performed at laboratory scale using four parallel filter columns. The periodic presence of O₃ (1-2 hours per week) at concentrations of 0.1-0.2 mg/L in the influent of an anthracite/sand filter had a negligible effect on the biological removals of acetate and formate. This was likely the result of the observed rapid decrease of the O₃ concentration in the top part of the filter. The periodic presence (1-2 hours per week) of H₂O₂ (～0.5 mg/L) in the influent of an anthracite/sand filter had no measurable effect on biological acetate and formate removals.     

熊果酸对血管内皮细胞增殖的影响及其机制

目的研究熊果酸(Ursolic acid,UA)对人脐静脉血管内皮细胞(Human umbilical vein endothelial cells,HUVECs)增殖及ERK1、c-Jun、c-Myc、Cyclin D1基因mRNA和蛋白表达的影响,探讨熊果酸抑制血管生长的机制。方法体外培养HU-VECs,用不同浓度熊果酸(31.5、62.5、125、250、500μg/ml)处理不同时间(12、24、48 h),MTT法检测细胞增殖抑制率。用125μg/ml熊果酸和100μmol/L PD98059(ERK抑制剂)处理HUVECs 48 h;不同浓度熊果酸处理24 h;125μg/ml熊果酸处理不同时间,RT-PCR和Western blot分别检测HUVECs中ERK1、c-Jun、c-Myc、Cyclin D1基因mRNA的转录水平和蛋白的表达水平。结果不同浓度的熊果酸对HUVECs的增殖均有明显的抑制作用,且呈剂量和时间依赖性;经熊果酸和PD98059处理的HUVECs中ERK1、c-Jun、c-Myc、Cyclin D1基因mRNA和蛋白的表达水平均明显降低,且呈剂量和时间依赖性。结论熊果酸可通过抑制ERK信号转导通路而抑制血管内皮细胞增殖。     

环氧油酸多元醇酯的制备与塑化PVC性能研究

使用季戊四醇和油酸通过酯化反应和环氧化反应制备了环保增塑剂环氧油酸多元醇酯。采用傅立叶变换红外光谱仪和核磁共振仪对制备的产品进行了表征。结果显示得到了预期增塑剂产品。将其作为主增塑剂与聚氯乙烯热塑共混,研究了塑化聚氯乙烯的最低共混扭矩、热性能、力学性能,对该增塑剂在不同介质中的耐迁移性进行了研究,并与邻苯类增塑剂和环氧大豆油的塑化性能进行了对比。研究发现,作为主增塑剂,环氧油酸多元醇酯增塑的聚氯乙烯(PVC)共混体系的热降解温度达到了281.5℃,拉伸强度和断裂伸长率分别为22.12 MPa和332.12%,在蒸馏水、乙酸溶液、乙醇溶液、石油醚和橄榄油中的迁移量明显低于邻苯二甲酸酯和环氧大豆油增塑的PVC共混体系。     

A Review on Current Strategies for Extraction and Purification of Hyaluronic Acid

Since it is known that hyaluronic acid contributes to soft tissue growth, elasticity, and scar reduction, different strategies of producing HA have been explored in order to satisfy the current demand of HA in pharmaceutical products and formulations. The current interest deals with production via bacterial and yeast fermentation and extraction from animal sources; however, the main challenge is the right extraction technique and strategy since the original sources (e.g., fermentation broth) represent a complex system containing a number of components and solutes, which complicates the achievement of high extraction rates and purity. This review sheds light on the main pathways for the production of HA, advantages, and disadvantages, along with the current efforts in extracting and purifying this high-added-value molecule from different sources. Particular emphasis has been placed on specific case studies attempting production and successful recovery. For such works, full details are given together with their relevant outcomes.