RNAi therapeutic and its innovative biotechnological evolution

Recently, United States Food and Drug Administration (FDA) and European Commission (EC) approved Alnylam Pharmaceuticals' RNA interference (RNAi) therapeutic, ONPATTRO? (Patisiran), for the treatment of the polyneuropathy of hereditary transthyretin-mediated (hATTR) amyloidosis in adults. This is the first RNAi therapeutic all over the world, as well as the first FDA-approved treatment for this indication. As a milestone event in RNAi pharmaceutical industry, it means, for the first time, people have broken through all development processes for RNAi drugs from research to clinic. With this achievement, RNAi approval may soar in the coming years. In this paper, we introduce the basic information of ONPATTRO and the properties of RNAi and nucleic acid therapeutics, update the clinical and preclinical development activities, review its complicated development history, summarize the key technologies of RNAi at early stage, and discuss the latest advances in delivery and modification technologies. It provides a comprehensive view and biotechnological insights of RNAi therapy for the broader audiences.     

茶树HMG-CoA还原酶基因全长cDNA克隆及序列分析

香气是茶叶的重要品质之一,萜类物质不仅香气好,而且沸点普遍较高,是构成茶叶香气的重要物质基础,决定着茶叶的香气品质,也可作为茶叶香型划分的依据。在植物中,倍半萜、多萜醇等通过胞质中的甲瓦龙酸(MVA)途径合成。HMG-Co A还原酶(HMGR)催化HMG-Co A(3-羟基-3-甲基戊二酸单酰辅酶A)生成甲瓦龙酸,是依赖MVA萜类合成途径的关键限速反应。为了有助于理解茶树萜类合成的分子遗传机制,通过RACE-PCR方法从茶树中克隆了一个编码HMG-Co A还原酶的c DNA全长序列(命名为Cs HMGR1),该序列由1 979 bp组成,包含一个1 722 bp的完整开放阅读框,编码573个氨基酸。其推定的编码蛋白与橡胶树、旱莲木、人参、荔枝、西洋参、丹参、罗汉果及龙眼的同源蛋白具有80%~82%的序列一致性。利用Cs HMGR1和其它物种HMGR同源蛋白的催化区域构建系统发育树,表明其属于真核生物I类HMGR家族。结构分析表明,Cs HMGR1含有两个跨膜区,推测其与其它真核生物同源蛋白类似地定位于内质网上;含有两个HMG-Co A结合位点、两个NADPH结合位点、四个保守的催化活性残基及一个磷酸化位点,说明磷酸化/去磷酸化很可能也是其活性调节的重要方式。表达分析表明,Cs HMGR1在"大叶龙"叶芽、母株叶芽及花芽都有较强的表达。其表达调控及生理活性对茶叶品质可能有重要影响,并在其功能解析的基础上,有可能作为茶叶品质鉴定及育种的一个依据。     

Multiple 100 Hz electroacupuncture treatments produced cumulative effect on the suppression of morphine withdrawal syndrome: Central preprodynorphin mRNA and p-CREB implicated

Alleviating opiate withdrawal syndrome in addicts is a critical precondition to break away from drug and further to prevent reuse. Electroacupuncture (EA) was claimed to be effective for alleviating withdrawal syndrome, but the optimal protocol remained unclear. In the present study we found that (1) 100 Hz EA administered 12-24 h after the last morphine injection suppressed the withdrawal syndrome in rats, multiple sessions of EA were more effective than single session, with the after-effect lasting for at least 7 days. (2) A down-regulation of preprodynorphin (PPD) mRNA level was observed in spinal cord, PAG and hypothalamus 60 h after the last morphine injection, which could be reversed by multiple sessions, but not a single session of EA. (3) Accompanied with the decrease of PPD mRNA level, there was an up-regulation of p-CREB in the three CNS regions, which was abolished by 100 Hz EA treatment. The findings suggest that down-regulation of p-CREB and acceleration of dynorphin synthesis in spinal cord, PAG and hypothalamus may be implicated in the cumulative effect of multiple 100 Hz EA treatment for opioid detoxification.     

Dynamic Changes of Tyrosine Hydroxylase and Dopamine Concentrations in the Ventral Tegmental Area-Nucleus Accumbens Projection During the Expression of Morphine-Induced Conditioned Place Preference in Rats

Our previous study demonstrated that morphine dose- and time-dependently elevated dopamine (DA) concentrations in the nucleus accumbens (NAc) during the expression of morphine-induced conditioned place preference (CPP) in rats. However, still unknown are how DA concentrations dynamically change during the morphine-induced CPP test and whether tyrosine hydroxylase (TH) activity in the ventral tegmental area (VTA) plays a vital role in this process. In the present study, we measured dynamic changes in TH and phosphorylated TH serine 40 (pTH Ser(40)) and pTH Ser(31) proteins in the VTA, and DA concentrations in the NAc at 5 min intervals during a 30 min morphine-induced CPP test. Rats that underwent morphine-induced CPP training significantly preferred the morphine-paired chamber during the CPP expression test, an effect that lasted at least 30 min in the drug-free state. DA concentrations in the NAc markedly increased at 15 min when the rats were returned to the CPP boxes to assess the expression of preference for the previously drug-paired chamber. DA concentrations then declined 2 h after the CPP test. TH and pTH Ser(40) levels, but not pTH Ser(31) levels, in the VTA were enhanced during the CPP test. These results indicated that TH and the phosphorylation of TH Ser(40) in the VTA may be responsible for DA synthesis and release in the NAc during the behavioral expression of conditioned reward elicited by a drug-associated context.     

半分子转运蛋白ABCG2的肿瘤多药耐药性研究及其应用

肿瘤常对临床上传统使用的多种化学治疗显示其内源性或获得性的药物耐受性即多药耐药性(multidrug resistance,MDR)．这种多药耐药性主要是由一类称为ABC(ATP-binding cassette)转运体蛋白超家族的跨膜蛋白引起的,它们结合并利用水解ATP提供的能量来转运药物,导致肿瘤细胞呈现抗药性．半分子转运蛋白ABCG2是近年来才发现的可归于ABC转运体大家族中的一个新成员,在肠、肝、胎盘和血脑屏障等部位大量表达,与全分子转运蛋白如P-gp (P-glycoprotein)和多药耐药蛋白(multi-drug resistance protein,MRP)相似,可以主动地把具有不同化学结构和作用于细胞内不同靶位点的化疗药物泵出胞外,从而引起肿瘤对多种抗癌药物(包括最新开发的药物)产生抗性．最近的一些十分有趣的研究还表明,ABCG2可能与干细胞分化状态和保护干细胞发育过程中免受周围环境的影响有关,而且还发现,它在侧群骨髓和神经干细胞内大量存在,因此,ABCG2可能在基因治疗中作为选择性的蛋白质标记正受到研究者们的极大关注．同时,ABCG2的单核苷酸多态性影响其结合并转运不同的底物/药物．鉴于ABCG2在肿瘤抗药性研究中的重要性以及它的一些新功能的初步研究表明,对ABCG2的生物学功能和作用机理以及在医学实践中的应用研究必将受到更大的关注．主要阐述了半分子ABC转运蛋白ABCG2的发现、重要的生化特性和生理功能及其相关的新研究进展和问题．     

黄芪注射液中对心脏正性肌力作用的成分分离与鉴定

用豚鼠离体乳头肌的收缩力相对增强百分率为正性肌力作用的指标,研究了黄芪注射液及其各分离部分的活性,证明中性部分为有效部位,其正性肌力作用呈浓度依赖关系,并用放射配基受体结合分析法,证明其作用不是通过β-肾上腺素受体,也不依赖于儿茶酚胺的释放,活性部分的成分,通过柱层析分离出单体,并经波谱数据分析和标准样品直接对照,证明该单体为黄芪甙IV。     

肺腺癌A549/DDP细胞周期变化及其多药耐药性

用Fura 2 /AM标记药物敏感的肺腺癌细胞A54 9和抗顺铂药物的肺腺癌细胞A54 9/DDP两种细胞胞内游离Ca2 ,用碘化丙锭 (PI)标记细胞DNA ,检测其胞内Ca2 的变化及两种细胞增殖能力和细胞周期 .实验结果表明 ,抗药性细胞株A54 9/DDP胞浆内游离Ca2 的浓度仅为药物敏感细胞株A54 9的 1/ 3左右 ,同时前者的细胞增殖能力较后者明显增强 ,而且细胞周期也明显缩短 .当用BAPTA AM和EGTA或A2 3187和Thapsigargin处理细胞以降低或升高其胞内自由Ca2 浓度时可改变细胞的生长周期 ,二者也呈现明显差别 .这些结果表明 ,对顺铂产生耐药性的人肺腺癌A54 9/DDP细胞胞内Ca2 浓度的降低 ,可能影响细胞的增殖 ,缩短细胞的生长周期 ,特别是影响起决定作用的G1期 ,从而有利于肿瘤细胞多药耐药特性的维持     

Gold nanoparticles: Emerging paradigm for targeted drug delivery system

The application of nanotechnology in medicine, known as nanomedicine, has introduced a plethora of nanoparticles of variable chemistry and design considerations for cancer diagnosis and treatment. One of the most important field is the design and development of pharmaceutical drugs, based on targeted drug delivery system (TDDS). Being inspired by physio-chemical properties of nanoparticles, TDDS are designed to safely reach their targets and specifically release their cargo at the site of disease for enhanced therapeutic effects, thereby increasing the drug tissue bioavailability. Nanoparticles have the advantage of targeting cancer by simply being accumulated and entrapped in cancer cells. However, even after rapid growth of nanotechnology in nanomedicine, designing an effective targeted drug delivery system is still a challenging task. In this review, we reveal the recent advances in drug delivery approach with a particular focus on gold nanoparticles. We seek to expound on how these nanomaterials communicate in the complex environment to reach the target site, and how to design the effective TDDS for complex environments and simultaneously monitor the toxicity on the basis of designing such delivery complexes. Hence, this review will shed light on the research, opportunities and challenges for engineering nanomaterials with cancer biology and medicine to develop effective TDDS for treatment of cancer.     

DB-02, a C-6-Cyclohexylmethyl Substituted Pyrimidinone HIV-1 Reverse Transcriptase Inhibitor with Nanomolar Activity,Displays an Improved Sensitivity against K103N or Y181C Than S-DABOs

6-(cyclohexylmethyl)-5-ethyl-2-((2-oxo-2-phenylethyl)thio)pyrimidin-4(3H)-one (DB-02) is a member of the newly reported synthetic anti-HIV-1 compounds dihydro-aryl/alkylsulfanyl-cyclohexylmethyl-oxopyrimidines, S-DACOs. In vitro anti-HIV-1 activity and resistance profile studies have suggested that DB-02 has very low cytotoxicity (CC₅₀>1mM) to cell lines and peripheral blood mononuclear cells (PBMCs). It displays potent anti-HIV-1 activity against laboratory adapted strains and primary isolated strains including different subtypes and tropism strains (EC₅₀s range from 2.40 to 41.8 nM). Studies on site-directed mutagenesis, genotypic resistance profiles revealed that V106A was the major resistance contributor for the compound. Molecular docking analysis showed that DB-02 located in the hydrophobic pocket with interactions of Lys101, Val106, Leu234, His235. DB-02 also showed non-antagonistic effects to four approved antiretroviral drugs. All studies indicated that DB-02 would be a potential NNRTI with low cytotoxicity and improved activity.