Antiviral activity and mechanism of action of novel thiourea containing chiral phosphonate on tobacco mosaic virus

Using half-leaf method O,O'-diisopropyl (3-(L-1-(benzylamino)-1-oxo-3- phenylpropan-2-yl)thioureido)(phenyl)methyl phosphonate (2009104) was studied for its activity on tobacco mosaic virus (TMV). It showed good curative activity in vivo and the curative activity at 500 μg/mL was found to be 53.3%. In vivo treatment with the control agent Ningnanmycin at 500 μg/mL resulted in 51.2% inhibition and curative inhibition rates respectively. Dot-ELISA test was employed to verify the efficacy of activity of compound 200910 for anti-TMV activity. The mechanism of action of compound 2009104 to resist TMV was also studied. The results showed that the resistance enzymes PAL, POD, SOD activity and chlorophyll content after TMV inoculation K(326) (Nicotiana tabacum K(326)) of tobacco plants followed by treatment with compound 2009104 were significantly enhanced. The study of the effect of compound 2009104 on TMV capsid protein (CP) showed that it inhibited the polymerization process of TMV-CP in vitro.     

Antofine analogues can inhibit tobacco mosaic virus assembly through small-molecule-RNA interactions

The extra unpaired base(s) or bulged structures of nucleic acids are capable either of forming complexes with nucleic-acid-binding proteins or of acting as binding sites for small molecules. We are interested in developing bulge-specific agents as potential drugs or chemical tools in biological research. Antofine can selectively bind with DNA and RNA bulged structures (Xi et al., Bioorg. Med. Chem. Lett. 2006, 16, 4300-4304). Furthermore, a series of antofine analogues suitable for selective binding with TMV RNA rather than with TMV coat protein (CP) were found. Biochemical studies indicated that antofine and its analogues disrupt in vitro virus assembly through small-molecule-RNA interactions. A structural model to illustrate these effects has been proposed. It is suggested that antofine analogues bind selectively with RNA bulged structures and therefore disrupt interaction between TMV RNA and TMV CP.     

Analysis of RNA phage fr coat protein assembly by insertion, deletion and substitution mutagenesis

A structure-function analysis of the icosahedral RNA bacteriophagefr coat protein (CP) assembly was undertaken using linker-insertion,deletion and substitution mutagenesis. Mutations were specificallyintroduced into either pre-existing or artificially createdrestriction enzyme sites within fr CP gene expressed in Escherichiacoli from a recombinant plasmid. This directs synthesis of wildtype protein that undergoes self-assembly and forms capsid-likeparticles indistinguishable morphologically and immunologicallyfrom native phage particles. A series of fr CP variants containingsequence alterations in the regions which are (i) exposed onthe external surface of capsid or (ii) located on the contactingareas between CP subunits were obtained and their assembly propertiesinvestigated. The majority of mutants demonstrated reductionof assembly ability and formed either CP dimers (mutations atresidues 2, 10, 63 or 129) or both dimer and capsid structures(residue 2 or 69). The exceptions were variants demonstratingnormal assembly and containing insertions at residues 2, 50or 129 of thefr CP. A third type of assembled structure wasformed by a variant with a single amino acid substitution I104T.The aA-helix region (residues 97-111) is particularly sensitiveto mutation and any alteration in this region decreases accumulationof mutant protein in E.coli. The relative contributions of particularfr CP domains in maintenance of capsid structural integrityas well as the possible capsid assembly mechanism are discussed.     

牛心朴子草抑制植物病毒活性成分安托芬的室内和田间活性测定

首次发现广泛分布于我国西北旱沙荒漠地带的牛心朴子草提取物中生物碱部位对危害极大的烟草花叶病毒具有很高的抑制活性,经生物活性跟踪与色谱分离,结构鉴定确认主要活性成分是安托芬。安托芬在1mg/L质量浓度下对烟草花叶病毒的抑制率高达60%。活体植株抗病毒活性的毒力测定结果表明,安托芬对活体植株烟草花叶病毒和活体植株马铃薯Y病毒具有很高的抑制活性,如安托芬在9mg/L的质量浓度下对活体植株烟草花叶病毒和活体植株马铃薯Y病毒的抑制率分别高达75%和80%。田间小区试验结果表明,牛心朴子草的效果明显好于两个对照药剂宁南霉素和病毒A,对烟草的生长发育无不良影响。     

Nanoparticle Encapsidation of Flock House Virus by Auto Assembly of Tobacco Mosaic Virus Coat Protein

Tobacco Mosaic virus (TMV) coat protein is well known for its ability to self-assemble into supramolecular nanoparticles, either as protein discs or as rods originating from the ~300 bp genomic RNA origin-of-assembly (OA). We have utilized TMV self-assembly characteristics to create a novel Flock House virus (FHV) RNA nanoparticle. FHV encodes a viral polymerase supporting autonomous replication of the FHV genome, which makes it an attractive candidate for viral transgene expression studies and targeted RNA delivery into host cells. However, FHV viral genome size is strictly limited by native FHV capsid. To determine if this packaging restriction could be eliminated, FHV was adapted to express enhanced green fluorescent protein (GFP), to allow for monitoring of functional FHV RNA activity. Then TMV OA was introduced in six 3'' insertion sites, with only site one supporting functional FHV GFP expression. To create nanoparticles, FHV GFP-OA modified genomic RNA was mixed in vitro with TMV coat protein and monitored for encapsidation by agarose electrophoresis and electron microscopy. The production of TMV-like rod shaped nanoparticles indicated that modified FHV RNA can be encapsidated by purified TMV coat protein by self-assembly. This is the first demonstration of replication-independent packaging of the FHV genome by protein self-assembly.     

共混纺丝组件用圆盘型静态混合器及模拟实验

从共混纺丝组件内部结构设计出发,根据静态混合器的混合原理,将组件内分配板设计成圆盘型静态混合器,每个分配圆盘中都设计出若干大小相等、径向均匀分布的菱形栅。通过复合纺丝模拟器的冷态模拟实验,分析模拟熔体在分配板流道内的流动混合状态。熔体经过菱形栅之间径向沟槽多次分割和汇集后,获得位置交换,完成多次的混合操作。结果表明,用于纺丝组件的圆盘型静态混合器能满足共混纺丝要求;将静态混合分配圆盘组配置在已有的纺丝组件内,其结构设计可行,混合效果显著。     

Modulating self-assembly of amyloidogenic proteins as a therapeutic approach for neurodegenerative diseases: strategies and mechanisms

Abnormal protein assembly causes multiple devastating disorders in the central nervous system (CNS), such as Alzheimer's, Parkinson's, Huntington's, and prion diseases. Due to the now extended human lifespan, these diseases have been increasing in prevalence, resulting in major public health problems and the associated financial difficulties worldwide. The wayward proteins that lead to disease self-associate into neurotoxic oligomers and go on to form fibrillar polymers through multiple pathways. Thus, a range of possible targets for pharmacotherapeutic intervention exists along these pathways. Many compounds have shown different levels of effectiveness in inhibiting aberrant self-assembly, dissociating existing aggregates, protecting cells against neurotoxic insults, and in some cases ameliorating disease symptoms in vivo, yet achieving efficient, disease-modifying therapy in humans remains a major unattained goal. To a large degree, this is because the mechanisms of action for these drugs are essentially unknown. For successful design of new effective drugs, it is crucial to elucidate the mechanistic details of their action, including the actual target(s) along the protein aggregation pathways, how the compounds modulate these pathways, and their effect at the cellular, tissue, organ, and organism level. Here, the current knowledge of major mechanisms by which some of the more extensively explored drug candidates work are discussed. In particular, we focus on three prominent strategies: 1)?stabilizing the native fold of amyloidogenic proteins, 2)?accelerating the aggregation pathways towards the fibrillar endpoint thereby reducing accumulation of toxic oligomers, and 3)?modulating the assembly process towards nontoxic oligomers/aggregates. The merit of each strategy is assessed, and the key points to consider when analyzing the efficacy of possible drug candidates and their mechanism of action are discussed.     

RNA干扰在害虫防治和杀虫剂靶标筛选中的应用前景

RNA干扰(RNA interference,RNAi)是生物体进化过程中抵御外源基因和病毒侵染的保守机制,在真核生物中存在的一种抗病毒入侵、抑制转座子活动和调控基因表达的监控机制.着重介绍了如何利用RNAi技术研究昆虫基因功能进展及利用这些功能基因的特异性沉默来进行害虫防治进展,并展望了RNAi技术防治害虫如何从实验室走向实践以及利用RNAi技术寻找新型杀虫剂的靶点和有活性化合物的毒理模型的前景.     

Platinum and Palladium Organometallic Compounds: Disrupting the Ergosterol Pathway in Trypanosoma cruzi

Current treatment for Chagas’ disease is based on two drugs, Nifurtimox and Benznidazol, which have limitations that reduce the effectiveness and continuity of treatment. Thus, there is an urgent need to develop new, safe and effective drugs. In previous work, two new metal-based compounds with trypanocidal activity, Pd-dppf-mpo and Pt-dppf-mpo, were fully characterized. To unravel the mechanism of action of these two analogous metal-based drugs, high-throughput omics studies were performed. A multimodal mechanism of action was postulated with several candidates as molecular targets. In this work, we validated the ergosterol biosynthesis pathway as a target for these compounds through the determination of sterol levels by HPLC in treated parasites. To understand the molecular level at which these compounds participate, two enzymes that met eligibility criteria at different levels were selected for further studies: phosphomevalonate kinase (PMK) and lanosterol 14-α demethylase (CYP51). Molecular docking processes were carried out to search for potential sites of interaction for both enzymes. To validate these candidates, a gain-of-function strategy was used through the generation of overexpressing PMK and CYP51 parasites. Results here presented confirm that the mechanism of action of Pd-dppf-mpo and Pt-dppf-mpo compounds involves the inhibition of both enzymes.     

轮丝链霉菌(Streptomyces Ys.03)代谢产物的抗植物病毒活性及其分类鉴定

[目的]1株从土壤中分离得到的放线菌的抗植物病毒活性及其分类鉴定。[方法]室内对峙培养、枯斑和系统寄主的保护及治疗试验,菌株形态观察并结合培养特征、生理生化性状及16S rDNA序列测定。[结果]菌株Ys.03对蜡状芽孢杆菌有稳定的拮抗作用;发酵液与烟草花叶病毒(TMV)混合后接种枯斑和系统寄主,对TMV抑制率分别为95.01%和88.67%;接种前、后叶面分别喷施发酵液抑制率均较高;菌株孢子链轮生,16S rDNA测序与师岗轮丝链霉菌(Streptomyces mormookaensis)同源性达100%。[结论]菌株Ys.03代谢产物对TMV有显著的抑制作用,鉴定为链霉菌属轮生类群师岗轮丝链霉菌。     

Enhancement of melting through corotating disk processors

Melting mechanisms in the corotating disk processor (DISK-PACK)

1 Farrel Company trademark.

are presented and discussed. This polymer processor consists of disk elements attached to a shaft with the entire assembly fitted into a housing. All polymer processes are induced by the drag action of the jointly moving disks, compared to only one moving surface characteristic of single screw extruders. Experiments carried out in a 150 and 7-1/2″ diameter corotating disk polymer processors indicate that polymers can be melted by either of two mechanisms: The drag melt removal mechanism and the dissipative mix-melting mechanism. The former mechanism occurs when a molten film is constantly removed from both sides of the solid bed, while the latter takes place throughout a system of molten and solid particulate mixture occupying the space between the disks. A theoretical model is presented for the drag melt removal (DMR) mechanism in such corotating disk polymer processors. The additional capability to induce a dissipative mix-melting (DMM) mechanism can increase the melting rate obtained from conventional mechanism by 300 percent and lower the exit melt temperature substantially. Melting rates over a broad range of conditions are presented for a variety of polymers.     

Synthesis and cytotoxicity of cyanoborane adducts of n6-benzoyladenine and 6-triphenylphosphonylpurine

N6-Benzoyladenine-cyanoborane (2), and 6-triphenylphosphonylpurine-cyanoborane (3) were selected for investigation of cytotoxicity in murine and human tumor cell lines, effects on human HL-60 leukemic metabolism and DNA strand scission to determine the feasibility of these compounds as clinical antineoplastic agents. Compounds 2 and 3 both showed effective cytotoxicity based on ED(50) values less than 4 mug/ml for L1210, P388, HL-60, Tmolt(3), HUT-78, HeLa-S(3) uterine, ileum HCT-8, and liver Hepe-2. Compound 2 had activity against ovary 1-A9, while compound 3 was only active against prostate PL and glioma UM. Neither compound was active against the growth of lung 549, breast MCF-7, osteosarcoma HSO, melanoma SK2, KB nasopharynx, and THP-1 acute monocytic leukemia. In mode of action studies in human leukemia HL-60 cells, both compounds demonstrated inhibition of DNA and protein syntheses after 60 min at 100 muM. These compounds inhibited RNA synthesis to a lesser extent. The utilization of the DNA template was suppressed by the compounds as determined by inhibition of the activities of DNA polymerase alpha, m-RNA polymerase, r-RNA polymerase and t-RNA polymerase, which would cause adequate inhibition of the synthesis of both DNA and RNA. Both compounds markedly inhibited dihydrofolate reductase activity, especially in compound 2. The compounds appeared to have caused cross-linking of the DNA strands after 24 hr at 100 muM in HL-60 cells, which was consistent with the observed increased in ct-DNA viscosity after 24 hr at 100 muM. The compounds had no inhibitory effects on DNA topoisomerase I and II activities or DNA-protein linked breaks. Neither compound interacted with the DNA molecule itself through alkylation of the nucleotide bases nor caused DNA interculation between base pairs. Overall, these antineoplastic agents caused reduction of DNA and protein replication, which would lead to killing of cancer cells.     

Binding of helix-threading peptides to E. coli 16S ribosomal RNA and inhibition of the S15-16S complex

Helix-threading peptides (HTPs) constitute a new class of small molecules that bind selectively to duplex RNA structures adjacent to helix defects and project peptide functionality into the dissimilar duplex grooves. To further explore and develop the capabilities of the HTP design for binding RNA selectively, we identified helix 22 of the prokaryotic ribosomal RNA 16S as a target. This helix is a component of the binding site for the ribosomal protein S15. In addition, the S15-16S RNA interaction is important for the ordered assembly of the bacterial ribosome. Here we present the synthesis and characterization of helix-threading peptides that bind selectively to helix 22 of E. coli 16S RNA. These compounds bind helix 22 by threading intercalation placing the N termini in the minor groove and the C termini in the major groove. Binding is dependent on the presence of a highly conserved purine-rich internal loop in the RNA, whereas removal of the loop minimally affects binding of the classical intercalators ethidium bromide and methidiumpropyl-EDTAFe (MPEFe). Moreover, binding selectivity translates into selective inhibition of formation of the S15-16S complex.     

Real-Time Fluorescence Microscopy on Living E. coli Sheds New Light on the Antibacterial Effects of the King Penguin β-Defensin AvBD103b

(1) Antimicrobial peptides (AMPs) are a promising alternative to conventional antibiotics. Among AMPs, the disulfide-rich β-defensin AvBD103b, whose antibacterial activities are not inhibited by salts contrary to most other β-defensins, is particularly appealing. Information about the mechanisms of action is mandatory for the development and approval of new drugs. However, data for non-membrane-disruptive AMPs such as β-defensins are scarce, thus they still remain poorly understood. (2) We used single-cell fluorescence imaging to monitor the effects of a β-defensin (namely AvBD103b) in real time, on living E. coli, and at the physiological concentration of salts. (3) We obtained key parameters to dissect the mechanism of action. The cascade of events, inferred from our precise timing of membrane permeabilization effects, associated with the timing of bacterial growth arrest, differs significantly from the other antimicrobial compounds that we previously studied in the same physiological conditions. Moreover, the AvBD103b mechanism does not involve significant stereo-selective interaction with any chiral partner, at any step of the process. (4) The results are consistent with the suggestion that after penetrating the outer membrane and the cytoplasmic membrane, AvBD103b interacts non-specifically with a variety of polyanionic targets, leading indirectly to cell death.     

Targeting RNA with small-molecule drugs: therapeutic promise and chemical challenges

Researchers' increasing awareness of the essential role played by RNA in many biological processes and in the progression of disease makes the discovery of new RNA targets an emerging field in drug discovery. Since most existing pharmacologically active compounds bind proteins, RNA provides nearly untapped opportunities for pharmacological development. The elucidation of the structure of the ribosome and other cellular and viral RNA motifs creates the opportunity for discovering new drug-like compounds that inhibit RNA function. However, further advances in understanding the chemistry and structure of RNA recognition are needed before these promises are fulfilled.     

Discovery of Potential Inhibitors for RNA-Dependent RNA Polymerase of Norovirus: Virtual Screening,and Molecular Dynamics

Noroviruses are non-enveloped viruses with a positive-sense single-stranded RNA (ssRNA) genome belonging to the genus Norovirus, from the family Caliciviridae, which are accountable for acute gastroenteritis in humans. The Norovirus genus is subdivided into seven genogroups, i.e., (GI-GVII); among these, the genogroup II and genotype 4 (GII.4) strains caused global outbreaks of human norovirus (HuNov) disease. The viral genome comprises three open reading frames (ORFs). ORF1 encodes the nonstructural polyprotein that is cleaved into six nonstructural proteins, which include 3C-like cysteine protease (3CLpro) and a viral RNA-dependent RNA polymerase. ORF2 and ORF3 encode the proteins VP1 and VP2. The RNA-dependent RNA polymerase (RdRp) from noroviruses is one of the multipurpose enzymes of RNA viruses vital for replicating and transcribing the viral genome, making the virally encoded enzyme one of the critical targets for the development of novel anti-norovirus agents. In the quest for a new antiviral agent that could combat HuNov, high throughput virtual screening (HTVS), combined with e-pharmacophore screening, was applied to screen compounds from the PubChem database. CMX521 molecule was selected as a prototype for a similarity search in the PubChem online database. Molecular dynamics simulations were employed to identify different compounds that may inhibit HuNov. The results predicted that compound CID-57930781 and CID-44396095 formed stable complexes with MNV-RdRp within 50 ns; hence, they may signify as promising human norovirus inhibitors.     

Drivers of soil net nitrogen mineralization in the temperate grasslands in Inner Mongolia,China

Soil net nitrogen mineralization (NNM) of four grasslands across the elevation and precipitation gradients was studied in situ in the upper 0–10 cm soil layer using the resin-core technique in Xilin River basin, Inner Mongolia, China during the growing season of 2006. The primary objectives were to examine variations of NNM among grassland types and the main influencing factors. These grasslands included Stipa baicalensis (SB), Aneulolepidum Chinense (AC), Stipa grandis (SG), and Stipa krylovii (SK) grassland. The results showed that the seasonal variation patterns of NNM were similar among the four grasslands, the rates of NNM and nitrification were highest from June to August, and lowest in September and October during the growing season. The rates of NNM and nitrification were affected significantly by the incubation time, and they were positively correlated with soil organic carbon content, total soil nitrogen (TN) content, soil temperature, and soil water content, but the rates of NNM and nitrification were negatively correlated with available N, and weakly correlated with soil pH and C:N ratio. The sequences of the daily mean rates of NNM and nitrification in the four grasslands during the growing season were AC > SG > SB > SK, and TN content maybe the main affecting factors which can be attributed to the land use type.     

Facile Fabrication of Electrohydrodynamic Micro‐/Nanostructures with High Aspect Ratio of a Conducting Polymer for Large‐Scale Superhydrophilic/Superhydrophobic Surfaces

This communication describes a straightforward, cost‐effective, and contactless lithography technique, electrohydrodynamic patterning, for fabricating the high‐aspect‐ratio micro‐/nanostructures of the conducting polymer (CP) films directly on a conductive substrate with high integrity and throughput. Meanwhile, the superhydrophilicity/superhydrophobicity of the final CP films can be obtained by modulating the ON/OFF‐state of the circuit of the assembly in the structure formation process. The final patterned CP films are hydrophilic/superhydrophilic when the circuit of the assembly is ON‐state in the structure formation process, while they are hydrophobic/superhydrophobic when the circuit of the assembly is OFF‐state. The high‐aspect‐ratio micro‐/nanostructures of the CP films with the superhydrophilic/superhydrophobic surface are important in both fundamental research and practical applications such as photovoltaics, sensors, supercapacitors, actuators, low friction surfaces, and water harvesting.