Abstract:自20世纪末RNA干扰现象(RNA interference)及其作用机制被发现以来，外源性的小干扰RNA(siRNA)已广泛地用于从基础研究到临床实践的多个领域。但，如何有效地、特异地将siRNA输送进入靶细胞，始终是使用者关注的重点，并已逐步成为siRNA应用于临床治疗的瓶颈问题之一。目前开展研究的siRNA转运方法主要包括3类：a. 通过与配基偶联实现siRNA的转运；b. 将siRNA包载于纳米颗粒等中经内吞进入细胞； c. 载体与细胞膜融合释放所载siRNA进入细胞。
本刊在这期中特意选择了3篇文章组成了一个小专题来介绍与探讨siRNA的输送问题。梁伟等应邀撰写了题为《siRNA脂质纳米输送载体的研究进展》的综述，介绍了siRNA输送载体的基本要求，特别是脂质纳米载体(lipid-based siRNA delivering systems)的设计和构筑原则，以及这类载体的研发现状和应用前景；张洪杰等结合自身的研发工作撰写了《细胞穿透肽及其结构改造在siRNA传递中的应用》一文，从发现、毒副作用、传递机制、结构修饰与传递功能改进等几个方面，系统评述了细胞穿透肽(cell penetrating peptides)在siRNA传递方面研究与应用的进展；张兴梅等在《适配子介导的siRNA转运》一文中重点介绍了基于适配子(aptamer)的siRNA转运系统的转运机制、近期研究进展和应用前景。3篇文章各有侧重，反映了当前siRNA转运研究中几个比较活跃的领域的研究进展，希望能对广大读者有所帮助。

Abstract:RNA interference (RNAi) is a specific gene-silencing mechanism triggered by small interfering RNA (siRNA). The application of RNAi in the clinic requires the development of safe and effective delivery systems. Efforts have been dedicated to the development of lipid-based systems in siRNA deliveries. Many of the lipid-based delivery vehicles' self-assemble with siRNA are through electrostatic interactions with charged amines. Electrostatic interactions must be stable enough to sustain the nucleic payload in the carrier en route, but must allow dissociation, to execute therapeutic activity, at the delivery site. Internalization of lipid-based siRNA delivery systems into cells typically occurs through endocytosis; accordingly, delivery requires materials that can facilitate endosomal escape. The size of the carrier is important as carriers <100 nm in diameter have been reported to have higher accumulation levels in tumours, hepatocytes and inflamed tissue. To reduce RES uptake and increase circulation time, carriers have been modified on the surface with polyethyleneglycol. Herein, we review basic requirements for building lipid-based siRNA delivery systems.

Abstract:The less of high efficient carriers to deliver siRNA drugs into their targeted tissues/cells with less toxicity are the major obstacles of siRNA pharmaceuticals in clinic application. The recent advance of siRNA delivery research based on cell-penetrating peptides (CPPs) in clinic opened the way of siRNA techniques for therapeutic application (Yi et al., Mol Ther (2011)19, 362-371). CPPs are short amphipathic and cationic peptides that are rapidly internalized across cell membranes. They can be used to deliver molecular cargos, such as imaging agents (fluorescent dyes and quantum dots), drugs, liposomes, peptide/protein, oligonucleotide/DNA/RNA, nanoparticles and bacteriophage into cells. The mechanism of cellular uptake and subsequent processing still remains controversial. It is now clear that CPP can mediate intracellular delivery via both endocytic and non-endocytic pathways. In this review, we discuss potential functions of CPPs, especially in structure modified CPPs for small RNA delivery in vitro and in vivo, highlighting their powerful promise for clinical efficacy.

Abstract:The ability of small interfering RNA (siRNA) to inhibit mammalian gene expression is being exploited as a new class of therapeutics for a variety of diseases．However, the efficient and safe delivery of siRNAs into specific cell populations is still the principal challenge in the clinical development of RNAi therapeutics．Many potential delivery vehicles and vectors have been explored including the aptamers targeting cell surface proteins．Selected nucleic acid binding species (aptamers) are high affinity and specificity for their targets, and they have been effectively applied in targeted therapy and diagnostics of diseases．The aptamer-based delivery of siRNAs can often enhance the therapeutic efficacy and reduce the unwanted off-target effects of siRNAs．Nowadays, some kinds of aptamers are able to mediate the delivery of siRNA, such as anti-PSMA aptamer, anti-gp120 aptamer. I will review the latest progress about aptamer mediated siRNA．

Abstract:A large quantity of literature highlights the interaction between tumor cells and the surrounding microenvironment which is a very important modulator of the processes in hepatocarcinogenesis. The microenvironment of hepatocellular carcinoma (HCC) can be obviously classified into cellular and produced non-cellular components, including hepatic stellate cells, tumor associated fibroblasts, immune cells and sinusoidal endothelial cells, extracellular matrix (ECM) proteins, growth factors and inflammatory cytokines which play an important role in the development and metastasis of HCC. In this review, we discuss the current cross-talk about the tumor microenvironment and tumor cells in pathogenesis of HCC.

Abstract:Cardiovascular disease is the leading cause of death and illness in developed countries today. Atherosclerosis is one of the most frequently ecountered diseases in angiocardiopathy. A number of studies have recently shown that PARP1 plays an important role in the mechanism of atherosclerosis. PARP1, a member of the PARP enzyme family, is an abundant nuclear protein which functions as a DNA nick-sensor enzyme. Poly(ADP-ribosylation) contributes to DNA repair and to the maintenance of genomic stability under normal circumstance. Overactivation of PARP consumes NAD⁺ and consequently ATP, culminating in cell dysfunction or necrosis. This cellular suicide mechanism has been implicated in the pathomechanism of atherosclerosis, myocardial ischemia, diabetes, and diabetes-associated cardiovascular dysfunction. Interestingly, several new and unexpected regulators of PARP1 activation have been found, including kinases, polyamines, caffeine metabolites, theophyline, and tetracycline antibiotics. The intracellular Ca²⁺ and NF-κB also participate in the regulatory mechanism. This review summarizes the biological function and general principle of targeted PARP, the possible pathomechanism of atherosclerosis, and the latest progress of PARP1-regulated atherosclerosis, which will make great strides in the study of atherosclerosis.

Abstract:Parkinson's disease (PD) is one of the most common neurodegenerative disorders. The causes of PD remain elusive, but mitochondrial malfunction is likely to be an important component. PD-related proteins PINK1 and Parkin affect mitochondrial function and morphology and participate in mitochondrial quality control. In Volume 147 Issue 4 of Cell (2011 Nov), Wang et al. published a paper entitled “PINK1 and Parkin target Miro for phosphorylation and degradation to arrest mitochondrial motility”. They identify the mitochondrial outer-membrane protein Miro as a phosphorylation substrate of PINK1 and show that Miro is degraded through a Parkin-denpendent mechanism after phosphorylation. Destruction of Miro unhooks damaged mitochondria from the microtubule network and prevents mitochondrial movement. The PINK1/Parkin pathway may quarantine damaged mitochondria prior to their clearance. This study not only reveals a glimpse into how PINK1 and Parkin cooperate, but also shows that mitochondrial quality control system can directly alter mitochondrial transport and raises the possibility that one cause of PD is the inappropriate transport of damaged mitochondria.

Abstract:The zinc-finger antiviral protein (ZAP) is a host factor that inhibits the replication of certain viruses, including murine leukemia virus and Sindbis virus by destabilizing viral mRNA in the cytoplasm. ZAP binds to specific viral mRNAs and recruits cellular RNA degradation machinery to degrade the RNA. Identifying ZAP-interacting proteins provides a viable strategy to uncover the mechanism by which ZAP inhibits viral replication. In the present study, we developed a method to search for proteins interacting with ZAP. Lysates of ZAP-expressing cells were subjected to glycerol gradient centrifugation. Fractions co-migrating with ZAP were collected, followed by immunoprecipitation of ZAP. Proteins co-immunoprecipitated with ZAP were identified by mass spectrometric analysis. By this method, PR65A, a structural subunit of PP2A, was identified as a putative ZAP-interacting protein. Coimmunoprecipitation assays confirmed the interaction between PR65A and ZAP in an RNA-independent manner. Downregulation of PR65A reduced the antiviral activity of ZAP. We conclude that PR65A interacts with ZAP and is required for the optimal antiviral activity of ZAP.

Abstract:The wall-associated kinases (WAKs) belong to a unique subfamily of the receptor-like kinases (RLKs) in plants, named for its tight association with the cell wall. There are 125 OsWAK genes in rice (Oryza sativa), of which OsWAK50 is a typical RLK with an extracellular domain, a transmembrane domain and an intracellular kinase domain. OsWAK50-GFP fusion protein is localized on the cell surface and associated with the cell wall. By the yeast two-hybrid screen, we identified 20 possible OsWAK50 intracellular interacting proteins. Further one to one reverse hybridization indicates that OsSK4, OsSWIB and OsSWI3C interact with OsWAK50 intracellular domain. OsSWIB interacts directly with the kinase domain of OsWAK50, while OsSK4 and OsSWI3C interact with OsWAK50 intracellular domain in a C terminal dependent manner. OsSK4 and OsSWIB can also interact with OsWAK53a, the closest homolog of OsWAK50 in rice, whereas OsSWI3C can not interact with OsWAK53a. The interaction between OsWAK50 and OsSK4, OsWAK53a and OsSK4 was further confirmed by in vivo BiFC assay. These results provide important clues for elucidating the molecular mechanisms of OsWAK50 function in rice.

Abstract:The in vivo treatment effect of CD (cytosine deaminase) suicidal genetic system on the glioma in rat was explored. The lentivirus carrier which was composed of CD genes was fabricated and the mouse BMMSCs were transfected by such CD genes to obtain constantly expressed cells, then BMMSCs were transplanted into the animal model which was built through an intracranial stereotactic inoculating method using a group of 40 SD rats. The rats were divided into 5 groups uniformly according to the cell type inoculated, i.e. ① C6 glioma, ②C6+MSCs(mesenchymal stems cells) cells (1∶1), ③C6+MSC cells (1∶2), ④C6+MSC -codA/eGFP cells (1∶1) and ⑤C6+MSC-codA/eGFP cells (1∶2). After 7 days of tumor formation, the abdominal cavity of rats were injected with 5-FC at 500 mg/(kg·d) for 14 days. Intensive scanning was carried out weekly to observe tumor volume using MRI. Simultaneously, survival times, routine pathological test, RT-PCR assay and HE staining were also operated. The results of MRI showed that the focus of infection in group ① presented round shape. The necrosis area of tumor found in the center, achieved an average volume of 246 mm³, and the survival time of the center area was 15.3 days. About the group ② and ③, the survival times were 16.0 and 16.6 days respectively. But in group ④ and ⑤, the survival times were both larger than 30 days, the necrosis area of tumor was 55 and 40 mm³ respectively at day 14, and the inhibition efficiency was 77.24% and 83.28% respectively after 28 days of treatment. It was concluded that the MRI scanning could clearly show the volume, shape, and internal structure of the tumor, which was highly related to the results of pathology. The treatment effect of bone marrow MSCs with CD genes and 5-FC therapy system on C6 intracranial glioma could be verified through dynamic MRI observation. Moreover, the RT-PCR test confirmed the expression of cytosine deaminase inside tumor organization.

Abstract:There is a growing concern about the relationship between the human health and the biological effects caused by the magnetic fields exposure. The cortical neurons isolated from the mice were exposed to 50 Hz magnetic fields (EMF 1 mT, 5 mT, 10 mT) for 15 min, and then the currents of the delayed rectifier potassium channel were recorded off-line using the whole-cell patch clamp technique to investigate the effects of EMF on channels for the first time. Compared to the control group, there was a significant inhibition on the I_k after exposure to EMF, and with the increase of the voltage depolarization, the inhibition rates of 1 mT and 5 mT almost unchanged and the inhibition rates were (30.0 ± 4.2)% and (20.0 ± 2.2)%，respectively. While the inhibition rate of 10 mT became larger and the maximum inhibition rate was 43.4%. Additionally, 1 mT and 5 mT magnetic fields both affected the activation characteristics of delayed rectifier potassium channel, the half activation voltage became larger and the slope factor unchanged, while 10 mT magnetic fields did not changed anything. This paper indicated that the structure and function of the channel protein on cell membrane may be altered by 50 Hz EMF, and there were different effects on the channel for different strength of magnetic fields, the window effects of strength of magnetic fields were improved in this study.

Abstract:There are two genes, fabI1 and fabI2 (UniProt AC: Q57A95 and Q57EU5), annotated as encodes putative enoyl-ACP reductase in Brucella abortus genome. Sequence alignment found that BaFabI1 and BaFabI2 are 50% and 51% identical to E. coli FabI, respectively. Further analysis identified that the catalytically active triad (Tyr-(Xaa)₆-Lys) of E. coli FabI are present in both BaFabI1 and BaFabI2. Expression of either of the two proteins restores the growth and the fatty acid synthesis of the E. coli fabI temperature sensitive mutant JP1111 under nonpermissive condition. In vitro assay identifies that both proteins restore the fatty acid synthetic ability and are active with substrates of all fatty acid chain lengths. These results demonstrated that B. abortus possesses two FabI-like enoyl-ACP reductases and it represents a new kind of diversity of bacterial enoyl-ACP reductase.

Abstract:β-Turn is a secondary protein structure type that is important in protein folding, protein stability and molecular recognition processes. To date, various methods have been put forward to predict β-turns, but none of them have tried directly to map the structures of pre-existing homologues from structural databases like RCSB PDB to the protein to be predicted. Given the large size of PDB (>70 000 structures), it is actually of high possibility to find a structural homologue for a newly identified sequence. In this work, we present a new method that predicts β-turns by combining homology information extracted from PDB with the results predicted by NetTurnP. Two datasets, the golden set BT426 and the self-constructed dataset EVA937, are used to assess our method. For each sequence in both datasets, only homologues deposited earlier than the sequence in PDB are employed. We have achieved Matthews correlation coefficients (MCCs) of 0.56, 0.52 respectively, which are higher than those obtained by NetTurnP alone of 0.50, 0.46, and the prediction accuracies (Q_total) obtained using our method are 81.4% and 80.4% separately, while NetTurnP alone achieves 78.2% and 77.3%. The results confirm that combining the homology information with state-of-the-art β-turn predictors like NetTurnP can significantly improve the prediction accuracy. A Java program called BTMapping has been written to implement our method, which is freely available at http://www.bio530.weebly.com together with the related datasets.

Abstract:MyD88 is an essential adaptor protein that mediates IL-1R/TLR signals. The homogeneous dimerization of MyD88 is required for its recruitment to the membrane receptors and is achieved by the interaction of its C-terminate TIR domain. After binding with the receptor, MyD88 dimer recruits the downstream molecules transmitting the inflammation signals and inducing gene expression. The present study aimed to establish a living-cell-fluorescence-based and high-through-put model for screening the inhibitors against the dimerization of MyD88 TIR. We constructed GFP-MyD88 TIR and RFP-MyD88 TIR plasmids, and transiently transfected them along with GFP or RFP into HeLa cells with different combinations. Under the excitation at 488 nm, the cells transfected with GFP-MyD88 TIR and RFP-MyD88 TIR plasmids showed the energy transfer from GFP to RFP, which is supposed to be mediated by the interaction of MyD88 TIR. Nevertheless, in the cells transfected with GFP-MyD88 TIR and RFP or RFP-MyD88 TIR and GFP plasmids, FRET was obviously impaired due to the absence of MyD88 TIR dimerization. The results suggest a possibility to establish a model for screening the inhibitors against the dimerization of MyD88 TIR: select the cell line that dually expresses GFP-MyD88 TIR and RFP-MyD88 TIR, and monitor the alteration of FRET upon the adding of different compounds in the medium. Additionally, we isolated and purified recombinant proteins His-MyD88 TIR and GST-MyD88 TIR, and performed in vitro protein binding assay. This recombinant protein binding assay can be used to verify the inhibitor candidates selected from the fluorescence screening model directly blocking the interaction of MyD88 TIR or not. The fluorescence screening model plus the in vitro verifying assay can be broadly used with commercially available compounds banks or lab-made natural products to find effective anti-inflammation inhibitors for therapeutic treatment of MyD88-pathway related chronic inflammation or autoimmune disorders.