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Research in nucleic acids has made major advances in the past decade in multiple fields of science and technology. Here we discuss some of the most important findings in DNA and RNA research in the fields of biology, chemistry, biotechnology, synthetic biology, nanostructures and optical materials, with emphasis on how chemistry has impacted, and is impacted by, these developments. Major challenges ahead include the development of new chemical strategies that allow synthetically modified nucleic acids to enter into, and function in, living systems., (This journal is © The Royal Society of Chemistry 2011)

The correct identification of the geographical origin of deer antlers is essential to quality control, as its positive physiological effects correlate with chemical components. In this study, we applied both genomics and metabolomics to the origin-identification of 101 samples from Canada, New Zealand, and Korea. The genomics identified deer species in each country but failed to categorize all the samples, due to the presence of identical species in different countries. For identical species, NMR-based metabolomics gave clean separations, compounds specific to each country were identified, and the validity was confirmed by prediction analysis. As the genomics provided unambiguous read-outs for different species, and the metabolomics cleanly distinguished among identical species from different countries, their combined use could be a robust method for origin-identification even in difficult cases. We believe the method to be generally applicable to many herbal medicinal products for which various species are grown internationally.

Background: Foot-and-mouth disease virus (FMDV) causes an economically important and highly contagious disease of cloven-hoofed animals. RNAi triggered by small RNA molecules, including siRNAs and miRNAs, offers a new approach for controlling viral infections. There is no report available for FMDV inhibition by vector-delivered miRNA, although miRNA is believed to have more potential than siRNA. In this study, the inhibitory effects of vector-delivered miRNAs targeting the 3D gene on FMDV replication were examined., Results: Four pairs of oligonucleotides encoding 3D-specific miRNA of FMDV were designed and selected for construction of miRNA expression plasmids. In the reporter assays, two of four miRNA expression plasmids were able to significantly silence the expression of 3D-GFP fusion proteins from the reporter plasmid, p3D-GFP, which was cotransfected with each miRNA expression plasmid. After detecting the silencing effects of the reporter genes, the inhibitory effects of FMDV replication were determined in the miRNA expression plasmid-transfected and FMDV-infected cells. Virus titration and real-time RT-PCR assays showed that the p3D715-miR and p3D983-miR plasmids were able to potently inhibit the replication of FMDV when BHK-21 cells were infected with FMDV., Conclusion: Our results indicated that vector-delivered miRNAs targeting the 3D gene efficiently inhibits FMDV replication in vitro. This finding provides evidence that miRNAs could be used as a potential tool against FMDV infection.

Hepatitis C virus is a major cause of chronic liver diseases which can lead to permanent liver damage, hepatocellular carcinoma and death. The presently available treatment with interferon plus ribavirin, has limited benefits due to adverse side effects such as anemia, depression and "flu-like" symptoms. Needless to mention, the effectiveness of interferon therapy is predominantly, if not exclusively, limited to virus type 3a and 3b whereas in Europe and North America the majority of viral type is 1a and 2a. Due to the limited efficiency of current therapy, RNA interference (RNAi) a novel regulatory and powerful silencing approach for molecular therapeutics through a sequence-specific RNA degradation process represents an alternative option. Several reports have indicated the efficiency and specificity of synthetic and vector based siRNAs inhibiting HCV replication. In the present review, we focused that combination of siRNAs against virus and host genes will be a better option to treat HCV.

Nonribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs) are large enzymes responsible for the biosynthesis of medically and ecologically important secondary metabolites. In a previous report, we described a proteomics approach to screen for expressed NRPSs or PKSs from bacteria with or without sequenced genomes. Here we used this proteome mining approach to discover a novel natural product arising from rare adenylation (A) and reductase (Red) domains in its biosynthetic machinery. We also cloned the entire gene cluster and elucidated the biosynthesis of the new compound, which is produced by an unsequenced Bacillus sp. isolated from soil collected in Koran, Louisiana., (© 2011 American Chemical Society)

Background: Marek's disease virus (MDV), which is widely considered to be a natural model of virus-induced lymphoma, has the potential to cause tremendous losses in the poultry industry. To investigate the structural basis of MDV membrane fusion and to identify new viral targets for inhibition, we examined the domains of the MDV glycoproteins gH and gB., Results: Four peptides derived from the MDV glycoprotein gH (gHH1, gHH2, gHH3, and gHH5) and one peptide derived from gB (gBH1) could efficiently inhibit plaque formation in primary chicken embryo fibroblast cells (CEFs) with 50% inhibitory concentrations (IC50) of below 12 μM. These peptides were also significantly able to reduce lesion formation on chorioallantoic membranes (CAMs) of infected chicken embryos at a concentration of 0.5 mM in 60 μl of solution. The HR2 peptide from Newcastle disease virus (NDVHR2) exerted effects on MDV specifically at the stage of virus entry (i.e., in a cell pre-treatment assay and an embryo co-treatment assay), suggesting cross-inhibitory effects of NDV HR2 on MDV infection. None of the peptides exhibited cytotoxic effects at the concentrations tested. Structural characteristics of the five peptides were examined further., Conclusions: The five MDV-derived peptides demonstrated potent antiviral activity, not only in plaque formation assays in vitro, but also in lesion formation assays in vivo. The present study examining the antiviral activity of these MDV peptides, which are useful as small-molecule antiviral inhibitors, provides information about the MDV entry mechanism.

Because the sugar moieties of natural products are primarily O-linked, the hydrolytic sensitivity of the glycosidic linkage limits their therapeutic application. One potential solution to this problem is to replace the labile O-glycosidic bond with an enzymatically and chemically stable C-glycosidic bond. In this study, computational analysis of the O-glycosyltransferase LanGT2 and the C-glycosyltransferase UrdGT2 was used to predict the changes necessary to switch the O-glycosylating enzyme to a C-glycosyltransferase. By screening rationally designed LanGT2 mutants a number of LanGT2 variants with C-glycosyltransferase activity were identified. One variant, having 10 amino acid substitutions, revealed the primary region that determines O- versus C-glycosylation. By modeling the active site of this mutant and probing the role of active site residues with alanine substitutions, this work also illuminates the mechanistic features of O- and C-glycosylation., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Ribosomal peptide natural products are ubiquitous, yet relatively few tools exist to predict structures and clone new pathways. Cyanobactin ribosomal peptides are found in ~30% of all cyanobacteria, but the connection between gene sequence and structure was not defined, limiting the rapid identification of new compounds and pathways. Here, we report discovery of four orphan cyanobactin gene clusters by genome mining and an additional pathway by targeted cloning, which represented a tyrosine O-prenylating biosynthetic pathway. Genome mining enabled discovery of five cyanobactins, including peptide natural products from Spirulina supplements. A phylogenetic model defined four cyanobactin genotypes, which explain the synthesis of multiple cyanobactin structural classes and help direct pathway cloning and structure prediction efforts. These strategies were applied to DNA isolated from a mixed cyanobacterial bloom containing cyanobactins., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

We report a synthetic biology strategy for rapid genetic manipulation of natural product biosynthetic pathways. Based on DNA assembler, this method synthesizes the entire expression vector containing the target biosynthetic pathway and the genetic elements required for DNA maintenance and replication in various hosts in a single-step manner through yeast homologous recombination, offering unprecedented flexibility and versatility in pathway manipulations.

In recent years, the number of known peptide natural products that are synthesized via the ribosomal pathway has rapidly grown. Taking advantage of sequence homology among genes encoding precursor peptides or biosynthetic proteins, in silico mining of genomes combined with molecular biology approaches has guided the discovery of a large number of new ribosomal natural products, including lantipeptides, cyanobactins, linear thiazole/oxazole-containing peptides, microviridins, lasso peptides, amatoxins, cyclotides, and conopeptides. In this review, we describe the strategies used for the identification of these ribosomally synthesized and posttranslationally modified peptides (RiPPs) and the structures of newly identified compounds. The increasing number of chemical entities and their remarkable structural and functional diversity may lead to novel pharmaceutical applications., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

The massive surge in genome sequencing projects has opened our eyes to the overlooked biosynthetic potential and metabolic diversity of microorganisms. While traditional approaches have been successful at identifying many useful therapeutic agents from these organisms, new tactics are needed in order to exploit their true biosynthetic potential. Several genomics-inspired strategies have been successful in unveiling new metabolites that were overlooked under standard fermentation and detection conditions. In addition, genome sequences have given us valuable insight for genetically engineering biosynthesis gene clusters that remain silent or are poorly expressed in the absence of a specific trigger. As more genome sequences are becoming available, we are noticing the emergence of underexplored or neglected organisms as alternative resources for new therapeutic agents., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Secondary metabolites from microorganisms have a broad spectrum of applications, particularly in therapeutics. The growing number of sequenced microbial genomes has revealed a remarkably large number of natural product biosynthetic clusters for which the products are still unknown. These cryptic clusters are potentially a treasure house of medically useful compounds. The recent development of new methodologies has made it possible to begin unlock this treasure house, to discover new natural products and to determine their biosynthesis pathways. This review will highlight some of the most recent strategies to activate silent biosynthetic gene clusters and to elucidate their corresponding products and pathways., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Cervi parvum cornu (CPC) is a well-known ethnopharmacological source, whereas Rangifer cornu (RC) is not considered to be a major source. CPC is distributed in sliced form. Addition of RC to CPC has become an issue in CPC distribution because the appearance of sliced RC is not different from sliced CPC. Therefore, a real-time polymerase chain reaction (PCR) method was developed in this study to detect contaminating RC in CPC. The C-VIC and R-FAM primer/probe sets were designed to specifically amplify CPC and RC DNA, respectively. The specificities and sensitivities of real-time PCR using two primer/probe sets and the applicability of the real-time PCR to powder mixtures, which involved mixtures of powdered CPC and powdered RC in diverse ratios, were evaluated. Real-time PCR using C-VIC and R-FAM primer/probe sets specifically and sensitively amplified both CPC and RC DNA. Furthermore, real-time RCR sensitively detected RC DNA in the powder mixtures of CPC and RC. These results indicate that this real-time PCR method using two primer/probe sets is sufficiently applicable for the detection of contaminant RC in CPC.

The remarkable potency and pharmacological diversity of animal venoms has made them an increasingly valuable source of lead molecules for drug and insecticide discovery. Nevertheless, most of the chemical diversity encoded within these venoms remains uncharacterized, despite decades of research, in part because of the small quantities of venom available. However, recent advances in the miniaturization of bioassays and improvements in the sensitivity of mass spectrometry and NMR spectroscopy have allowed unprecedented access to the molecular diversity of animal venoms. Here, we discuss these technological developments in the context of establishing a high-throughput pipeline for venoms-based drug discovery.

Paraoxonase (PON)-1 is the most potent human organophosphatase known, but recombinant forms of human PON1 have been difficult to produce owing to poor secretion by host cells. In the present investigation, human PON1 is re-engineered as an IgG-PON1 fusion protein. The 355 amino acid human PON1 is fused to the carboxyl terminus of the heavy chain of a chimeric monoclonal antibody (MAb) against the human insulin receptor (HIR), and this fusion protein is designated HIRMAb-PON1. The HIRMAb part of the fusion protein enables brain penetration of the PON1, which was considered important, because organophosphate toxicity causes death via a central nervous system site of action. A high producing line of stably transfected Chinese hamster ovary (CHO) cells secreting the HIRMAb-PON1 fusion protein in the absence of serum or lipid acceptors was cloned. The bioreactor generated fusion protein was purified to homogeneity with low impurities by protein A affinity chromatography and anion exchange chromatography. The HIRMAb-PON1 fusion protein was stable as a sterile liquid formulation stored at 4°C for at least 1 year. The plasma pharmacokinetics (PK) of the HIRMAb-PON1 fusion protein was evaluated in Rhesus monkeys, which is the first PK evaluation of a recombinant PON1 protein. The fusion protein was rapidly removed from blood, primarily by the liver. The blood-brain barrier permeation of the HIRMAb-PON1 fusion protein was high and comparable to other HIRMAb fusion proteins. Re-engineering human PON1 as the HIRMAb fusion protein allows for production of a stable, field-deployable formulation of the enzyme that is brain-penetrating., (© 2010 Wiley Periodicals, Inc.)

Interferons (IFNs) are a first line of defense against viral infection. Herein we describe the use of an adenovirus vectored mouse IFN alpha gene (mDEF201) as a prophylactic and treatment countermeasure in a SARS-CoV-infected BALB/c mouse model. Complete survival protection was observed in mice given a single dose of mDEF201 administered intranasally 1, 3, 5, 7, or 14 days prior to lethal SARS-CoV challenge (p<0.001), and body weights of these treated mice were unaffected by the challenge. In addition, low doses of mDEF201 protected lungs in a dose dependent manner as measured by a reduction in gross pathology. Intranasal treatment with mDEF201 ranging from 10(6) to 10(8)PFU significantly protected mice against a lethal SARS-CoV infection in a dose dependent manner up to 12h post infection (p<0.001). The data suggest that mDEF201 is a new class of antiviral agent further development as treatment for SARS-CoV infections., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Murine leukemia virus (MLV)-based retroviral vector is widely used for gene transfer. Efficient packaging of the genomic RNA is critical for production of high-titer virus. Here, we report that expression of the insulin-like growth factor II mRNA binding protein 1 (IMP1) enhanced the production of infectious MLV vector. Overexpression of IMP1 increased the stability of viral genomic RNA in virus producer cells and packaging of the RNA into progeny virus in a dose-dependent manner. Downregulation of IMP1 in virus producer cells resulted in reduced production of the retroviral vector. These results indicate that IMP1 plays a role in regulating the packaging of MLV genomic RNA and can be used for improving production of retroviral vectors.

In this study, a standard strain of HSV-1 (strain SM(44)) was used to investigate the antiviral activity of the recombinant Cyanovirin-N (CV-N) against Herpes simplex virus type 1 (HSV-1) in vitro and in vivo. Cytopathic effect (CPE) and MTT assays were used to evaluate the effect of CV-N on HSV-1 in Vero cells. The number of copies of HSV-DNA was detected by real-time fluorescence quantitative PCR (FQ-PCR). The results showed that CV-N had a low cytotoxicity on Vero cells with a CC(50) of 359.03 ± 0.56 μg/mL, and that it could not directly inactivate HSV-1 infectivity. CV-N not only reduced the CPE of HSV-1 when added before or after viral infection, with a 50% inhibitory concentration (IC(50)) with 2.26 and 30.16 μg/mL respectively, but it also decreased the copies of HSV-1 DNA in infected host cells. The encephalitis model for HSV-1 infection was conducted in Kunming mice, and treated with three dosages of CV-N (0.5, 5 & 10 mg/kg) which was administered intraperitoneally at 2h, 3d, 5d, 7d post infection. The duration for the appearance of symptoms of encephalitis and the survival days were recorded and brain tissue samples were obtained for pathological examination (HE staining). Compared with the untreated control group, in the 5mg/kg CV-N and 10mg/kg CV-N treated groups, the mice suffered light symptoms and the number of survival days were more than 9 d and 14 d respectively. HE staining also showed that in 5mg/kg CV-N and 10mg/kg CV-N treated groups, the brain cells did not show visible changes, except for a slight inflammation. Our results demonstrated that CV-N has pronounced antiviral activity against HSV-1 both in vitro and in vivo, and it would be a promising new candidate for anti-HSV therapeutics.

Halogenation, which was once considered a rare occurrence in nature, has now been observed in many natural product biosynthetic pathways. However, only a small fraction of halogenated compounds have been isolated from terrestrial plants. Given the impact that halogenation can have on the biological activity of natural products, we reasoned that the introduction of halides into medicinal plant metabolism would provide the opportunity to rationally bioengineer a broad variety of novel plant products with altered, and perhaps improved, pharmacological properties. Here we report that chlorination biosynthetic machinery from soil bacteria can be successfully introduced into the medicinal plant Catharanthus roseus (Madagascar periwinkle). These prokaryotic halogenases function within the context of the plant cell to generate chlorinated tryptophan, which is then shuttled into monoterpene indole alkaloid metabolism to yield chlorinated alkaloids. A new functional group-a halide-is thereby introduced into the complex metabolism of C. roseus, and is incorporated in a predictable and regioselective manner onto the plant alkaloid products. Medicinal plants, despite their genetic and developmental complexity, therefore seem to be a viable platform for synthetic biology efforts.

Background: Hepatitis C virus (HCV) is a major causative agent of liver associated diseases throughout the world, with genotype 3a responsible for most of the cases in Pakistan. Due to the limited efficiency of current therapy, RNA interference (RNAi) a novel regulatory and powerful silencing approach for molecular therapeutics through a sequence-specific RNA degradation process represents an alternative option., Results: The current study was purposed to assess and explore the possibility of RNAi to silence the HCV-3a Core gene expression, which play complex role in regulation of cell growth and host genes expression essential for infectivity and disease progression. To identify the potent siRNA target sites, 5 small interfering RNAs (siRNAs) against Core gene were designed and in vitro transcribed after consensus sequence analysis of different HCV-3a isolates. Antiviral effects of siRNAs showed upto 80% inhibition of Core gene expression by different siRNAs into Huh-7 cells as compared with Mock transfected and control siRNAs treated cells. For long lasting effect of siRNAs, vector based short hairpin siRNAs (shRNAs) were designed and tested against HCV-3a Core which resulted in a similar pattern of inhibition on RNA and protein expression of HCV Core as synthetic siRNAs. Furthermore, the efficacy of cell culture tested siRNA and shRNA, were evaluated for inhibition of HCV replication in HCV infected serum inoculated Huh-7 cells and a significant decrease in HCV viral copy number was observed., Conclusions: Our results support the possibility of using consensus siRNA and shRNA-based molecular therapy as a promising strategy in effective inhibition of HCV-3a genotype.

A detailed bioinformatics analysis of six glycopeptide biosynthetic gene clusters isolated from soil environmental DNA (eDNA) megalibraries indicates that a subset of these gene clusters contains collections of tailoring enzymes that are predicted to result in the production of new glycopeptide congeners. In particular, sulfotransferases appear in eDNA-derived gene clusters at a much higher frequency than would be predicted from the characterization of glycopeptides from cultured Actinomycetes . Enzymes found on tailoring-enzyme-rich eDNA clones associated with these six gene clusters were used to produce a series of new sulfated glycopeptide derivatives in both in vitro and in vivo derivatization studies. The derivatization of known natural products with eDNA-derived tailoring enzymes is likely to be a broadly applicable strategy for generating libraries of new natural product variants.

In the recent years, the number of drug- and multi-drug-resistant microbial strains has increased rapidly. Therefore, the need to identify innovative approaches for development of novel anti-infectives and new therapeutic targets is of high priority in global health care. The detection of small RNAs (sRNAs) in bacteria has attracted considerable attention as an emerging class of new gene expression regulators. Several experimental technologies to predict sRNA have been established for the Gram-negative model organism Escherichia coli. In many respects, sRNA screens in this model system have set a blueprint for the global and functional identification of sRNAs for Gram-positive microbes, but the functional role of sRNAs in colonization and pathogenicity for Listeria monocytogenes, Staphylococcus aureus, Streptococcus pyogenes, Enterococcus faecalis and Clostridium difficile is almost completely unknown. Here, we report the current knowledge about the sRNAs of these socioeconomically relevant Gram-positive pathogens, overview the state-of-the-art high-throughput sRNA screening methods and summarize bioinformatics approaches for genome-wide sRNA identification and target prediction. Finally, we discuss the use of modified peptide nucleic acids (PNAs) as a novel tool to inactivate potential sRNA and their applications in rapid and specific detection of pathogenic bacteria., (© 2010 The Authors. Journal compilation © 2010 Society for Applied Microbiology and Blackwell Publishing Ltd.)

Small interfering RNAs (siRNAs) have been shown to effectively downregulate gene expression in human cells, giving them potential to eradicate disease. Prospects for clinical applications are discussed in this review, along with an overview of recent history and our current understanding of siRNAs used for therapeutic application in human diseases, such as cancer and viral infections. Over recent years, progress has been made in lipids, ligands, nanoparticles, polymers and viral vectors as delivery agents and for gene-based expression of siRNA to enhance the efficacy and specificity of these methods while at the same time reducing toxicity. It has become apparent that given the recent advances in chemistry and delivery, RNAi will soon prove to be an important and widely used therapeutic modality., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Angiogenesis is important in tumor development. Vascular endothelial growth factor (VEGF) is involved in this process. In this report, we constructed a recombinant protein (called FK) by fusing the second immunoglobulin-like (Ig-like) domain of a human fms-like tyrosine kinase (Flt-1) with the third Ig-like domain of human kinase insert domain-containing receptor (KDR). FK bound to VEGF(165) in a dose-dependent manner with a disocciation constant (Kd) of 2.7 pM. In addition, FK specifically inhibited the proliferation of human microvascular endothelial cell (HMEC) and human umbilical vein endothelial Cell (HUVEC) stimulated by VEGF(165). Subsequent studies also demonstrate that FK efficaciously suppresses growth of a variety of tumors, which could make FK a potential drug candidate in anti-tumor therapy.

Transplantation of mesenchymal stem cells (MSC) improves repair and function recovery following myocardial infarction (MI), but underlying mechanisms remain to be elucidated. We hypothesize that MSC could achieve protection by paracrine effects through released mediators rather than direct cardiac regeneration. We sought to characterize the effects of MSC-secreted growth factors on extent of early recovery from MI. Swine subjected to acute MI by temporary balloon occlusion of the left anterior descending coronary artery using percutaneous techniques received intracoronary injection of either concentrated MSC-derived growth factors or control medium. Animals were killed at 7 days to evaluate early effects. Treatment with MSC-derived factors significantly reduced cardiac troponin-T elevation and improved echocardiographic parameters, including fractional area shortening, stroke volume, cardiac output, and wall motion score index. Quantitative evaluation of fibrosis by Verhoff staining revealed a reduction of the fibrotic area in the infarcted zone. Similarly, Masson's trichrome staining revealed reduced myocardial damage as demonstrated by areas of relatively preserved myocardium in the infarcted area. TUNEL assay demonstrated less cardiomyocyte apoptosis. Protein array detected the presence of angiogenic (vascular endothelial growth factor, endothelin, and epiregulin), anti-apoptotic (Galectin-3, Smad-5, sRFP-1, and sRFP-4) and anti-remodeling factors. Reverse transcription polymerase chain reaction confirmed the expression of these factors. In summary, a single intracoronary injection of concentrated biologically active factors secreted by MSC could achieve early protection of ischemic myocardium and improve cardiac repair and contractility. MSC-derived growth factors injection (rather than MSC themselves) should be evaluated as a novel therapy to treat ischemic heart disease, avoiding many practical and technical issues of cell therapy.

A54145 factors are calcium-dependent lipopeptide antibiotics produced by Streptomyces fradiae NRRL 18160. A54145 is structurally related to the clinically important daptomycin, and as such may be a useful scaffold for the development of a novel lipopeptide antibiotic. We developed methods to genetically manipulate S. fradiae by deletion mutagenesis and conjugal transfer of plasmids from Escherichia coli. Cloning the complete pathway on a bacterial artificial chromosome (BAC) vector and the construction of ectopic trans-complementation with plasmids utilizing the φC31 or φBT1 site-specific integration system allowed manipulation of A54145 biosynthesis. The BAC clone pDA2002 was shown to harbor the complete A54145 biosynthesis gene cluster by heterologous expression in Streptomyces ambofaciens and Streptomyces roseosporus strains in yields of >100 mg/liter. S. fradiae mutants defective in LptI methyltransferase function were constructed, and they produced only A54145 factors containing glutamic acid (Glu₁₂), at the expense of factors containing 3-methyl-glutamic acid (3mGlu₁₂). This provided a practical route to produce high levels of pure Glu₁₂-containing lipopeptides. A suite of mutant strains and plasmids was created for combinatorial biosynthesis efforts focused on modifying the A54145 peptide backbone to generate a compound with daptomycin antibacterial activity and activity in Streptococcus pneumoniae pulmonary infections.

Despite significant efforts in many countries, there is still no commercially viable dengue vaccine. Currently, attention is focused on the development of either live attenuated vaccines or live attenuated chimaeric vaccines using a variety of backbones. Alternate vaccine approaches, such as whole inactivated virus and subunit vaccines are in the early stages of development, and are each associated with different problems. Subunit vaccines offer the advantage of providing a uniform antigen of well-defined nature, without the added risk of introducing any genetic material into the person being inoculated. Preliminary trials of subunit vaccines (using dengue E protein) in rhesus monkeys have shown promising results. However, the primary disadvantages of dengue subunit vaccines are the low levels of expression of dengue proteins in mammalian or insect cells, as well as the added unknown risks of antigens produced from mammalian cells containing other potential sources of contamination. In the past two decades, plants have emerged as an alternative platform for expression of biopharmaceutical products, including antigens of bacterial, fungal or viral origin. In the present minireview, we highlight the current plant expression technologies used for expression of biopharmaceutical products, with an emphasis on plants as a production system for dengue subunit vaccines.

Recent work has shown that it is possible to target regulatory elements to DNA sequences of an investigator's choosing, increasing the armamentarium for probing gene function. In this review, we discuss the development and use of designer zinc finger proteins (ZFPs) as sequence specific tools. While the main focus of this review is to discuss the attachment of the FokI nuclease to ZFPs and the ability of the resulting fusion protein (termed zinc finger nucleases (ZFNs)) to genomically manipulate a gene of interest, we will also cover the utility of other functional domains, such as transcriptional activators and repressors, and highlight how these are being used as discovery and therapeutic tools.

The nucleocapsids formation is a pivotal step of hepatitis B virus (HBV) life cycle. The inhibition of HBV nucleocapsids assembly is a promising strategy for the anti-HBV treatment. HBc78-117 is an internal fragment of hepatitis B core protein (HBc). In this study, we used lentiviral vector to deliver HBc78-117 cDNA sequence into HepG2.2.15 cells and examined the effect of HBc78-117 on HBV replication. We confirmed by immunoprecipitation analysis that HBc78-117 interacted with full-length HBc in HepG2.2.15 cells. The nucleocapsids and HBV DNA replication intermediates were markedly reduced in the cells expressing HBc78-117, although HBV pregenome RNA was not affected. The level of HBV DNA was also significantly reduced in culture supernatant. These suggest that HBc78-117 can inhibit HBV DNA replication by interfering with nucleocapsids assembly., ((c) 2010 Elsevier B.V. All rights reserved.)

RNA silencing has been adopted to develop virus-resistant plants through expression of virus-derived hairpin RNAs. Due to the high sequence specificity of RNA silencing, this technology has been limited to the targeting of single viruses. Simultaneous targeting of multiple viruses or plant genes can be achieved by using a chimeric cassette. In this study, a simple method was developed to construct chimeric hairpin RNA rapidly and efficiently. This method splices two DNA fragments from viruses or plant genes to be a chimeric sequence using Overlap Extension PCR (OE-PCR); then this chimeric sequence was assembled with an intron sequence to generate an intron-containing hairpin RNA construct in one step mediated by OE-PCR. This method is neither dependent on restriction enzymes nor requires expensive consumables, so a chimeric hairpin RNA can be constructed rapidly and costlessly. Two chimeric hairpin RNA constructs were amplified successfully using this method, with the targeting sequences from both papaya ringspot virus (PRSV) and two plant genes encoding translation initiation factors eIF4E and eIFiso4E. This novel method is a useful strategy to construct chimeric hairpin RNA for RNA silencing in plants., (Copyright 2010 Elsevier B.V. All rights reserved.)

Gene silencing by RNA interference (RNAi) can be achieved by intracellular expression of a short hairpin RNA (shRNA) that is processed into the effective small interfering RNA (siRNA) inhibitor by the RNAi machinery. Previous studies indicate that shRNA molecules do not always reflect the activity of corresponding synthetic siRNAs that attack the same target sequence. One obvious difference between these two effector molecules is the hairpin loop of the shRNA. Most studies use the original shRNA design of the pSuper system, but no extensive study regarding optimization of the shRNA loop sequence has been performed. We tested the impact of different hairpin loop sequences, varying in size and structure, on the activity of a set of shRNAs targeting HIV-1. We were able to transform weak inhibitors into intermediate or even strong shRNA inhibitors by replacing the loop sequence. We demonstrate that the efficacy of these optimized shRNA inhibitors is improved significantly in different cell types due to increased siRNA production. These results indicate that the loop sequence is an essential part of the shRNA design. The optimized shRNA loop sequence is generally applicable for RNAi knockdown studies, and will allow us to develop a more potent gene therapy against HIV-1.

Synaptotagmin II (syt II) is the specific protein receptor of botulinum neurotoxin B (BoNT/B), and the luminal domain of syt II contains toxin-binding sites that have a high affinity for BoNT/B. However, it is not yet clear whether the luminal domain of syt II (syt II-LD) inhibits the toxicity of BoNT/B by interfering with the toxin-receptor interaction. In this study, we characterized the binding of the purified recombinant syt II-LD to BoNT and revealed that the recombinant syt II-LD in vivo could provide protection against BoNT/B intoxication in mice models, and the neutralization effect could be improved by using gangliosides., (Copyright 2010 Elsevier Masson SAS. All rights reserved.)

The review highlights the recent progress on the elucidation of the biosynthetic mechanism of enediyne natural products. A large body of knowledge has been gathered from the genetic, biochemical and structural studies since the sequencing of the first two enediyne gene clusters in 2002. Primary attention is devoted to the novel pathways and enzymes in the biosynthesis of the enediyne warhead as well as the peripheral moieties.

Transmissible gastroenteritis virus (TGEV) is the causative agent of porcine transmissible gastroenteritis (TGE), characterized by high mortality and severely retarded growth in piglets that dramatically affects the porcine industry. Previously, we have identified two shRNA-expressing plasmids pEGFP-U6/P1 and pEGFP-U6/P2 that target RNA-dependent RNA polymerase (RdRP) gene of TGEV with more than 95% of virus inhibition in vitro. In this study, inhibition of the TGEV replication by pEGFP-U6/P1 and pEGFP-U6/P2 was tested in mini-pigs. SPF mini-pigs at 25 days old were injected with the shRNA-expressing plasmids and then infected with TGEV. The results from the analyses of clinical signs, histopathology, indirect immunofluorescence (IIF) and RT-PCR show that the two shRNA-expressing plasmids could significantly decrease the quantity of TGEV in different organs and protect mini-pigs from TGEV infection. These findings illustrate the prospect for TGEV-specific shRNAs to be new anti-TGEV agents., (Crown Copyright 2010. Published by Elsevier B.V. All rights reserved.)

Natural products have played a prominent role in the history of organic chemistry, and they continue to be important as drugs, biological probes, and targets of study for synthetic and analytical chemists. In this Perspective, we explore how connecting Nature's small molecules to the genes that encode them has sparked a renaissance in natural product research, focusing primarily on the biosynthesis of polyketides and non-ribosomal peptides. We survey monomer biogenesis, coupling chemistries from templated and non-templated pathways, and the broad set of tailoring reactions and hybrid pathways that give rise to the diverse scaffolds and functionalization patterns of natural products. We conclude by considering two questions: What would it take to find all natural product scaffolds? What kind of scientists will be studying natural products in the future?

Hepatitis E virus (HEV) is a non-enveloped, single-stranded, positive sense RNA virus, which is a major cause of water-borne hepatitis. RNA interference (RNAi) is a sequence-specific cellular antiviral defence mechanism, induced by double-stranded RNA, which we used to investigate knockdown of several genes and the 3' cis-acting element (CAE) of HEV. In the present report, shRNAs were developed against the putative helicase and replicase domains and the 3'CAE region of HEV. Production of siRNA was confirmed by northern hybridization. The possible innate response induction due to shRNA expressions was verified by transcript analysis for interferon-beta and 2',5'-oligoadenylate synthetase genes and was found to be absent. Initially, the selected shRNAs were tested for their efficiency against the respective genes/3'CAE using inhibition of fused viral subgenomic target domain-renilla luciferase reporter constructs. The effective shRNAs were studied for their inhibitory effects on HEV replication in HepG2 cells using HEV replicon and reporter replicon. RNAi mediated silencing was demonstrated by reduction of luciferase activity in subgenomic target-reporter constructs and reporter replicon. The real time PCR was used to demonstrate inhibition of native replicon replication in transfected cells. Designed shRNAs were found to be effective in inhibiting virus replication to a variable extent (45-93%).

L-methioninase is ubiquitous in all organisms except in mammals. It mainly catalyzes the, alpha, gamma-elimination of L: -methionine to alpha-ketobutyrate, methanethiol, and ammonia. Unlike normal cells, methionine dependency was reported as a biochemical phenomenon among various types of cancer cells. Thus, L-methioninase is the universal protocol for triggering the majority of tumor cells. This review is an attempt to briefly describe the occurrence of the biochemical and molecular properties of L-methioninase by a comparative manner to the eukaryotic and prokaryotic source for the maximum exploitation in the therapeutic field. The combination of L-methioninase treatment, gene therapy, and chemotherapeutic drugs clearly explores the various therapeutic aspects of this enzyme. Finally, the perspectives for increasing the therapeutic efficacy of this enzyme were described.

Background and Aim: The intestinal epithelium is constantly exposed to high levels of genetic material like bacterial DNA. Under normal physiological conditions, the intestinal epithelial monolayer as a formidable dynamic barrier with a high-polarity structure facilitates only a controlled and selective flux on components between the lumen and the underlining mucosa and even is able to facilitate structure-based macromolecules movement. The aim of this study was to test the effect of natural commensal-origin DNA on the TLR9 signaling cascade and the barrier integrity of polarized intestinal epithelial cells (IECs)., Methods: : Polarized HT-29 and T84 cells were treated with TNF-alpha in the presence or absence of DNA from Lactobacillus rhamnosus GG (LGG) and Bifidobacterium longum. TLR9 and interleukin-8 (IL-8) mRNA expression was assessed by semiquantitative and TaqMan real-time reverse-transcription polymerase chain reaction. Expression of TLR9 protein, degradation of inhibitor of kappa B alpha (IkappaBalpha), and p38 mitogen-activated protein kinase (p38 MAP) phosphorylation were assessed by Western blotting. To further reveal the role of TLR9 signaling, the TLR9 gene was silenced by siRNA. IL-8 secretion was measured by an enzyme-linked immunosorbent assay. Nuclear factor-kappa B (NF-kappaB) activity was assessed by the electrophoretic mobility shift assay (EMSA) and NF-kappaB-dependent luciferase reporter gene assays. As an indicator of tight junction formation and monolayer integrity of epithelial cell monolayers, transepithelial electrical resistance (TER) was repetitively monitored. Transmonolayer movement of natural commensal-origin DNA across monolayers was monitored using qRT-PCR and nested PCR based on bacterial 16S rRNA genes., Results: In response to apically applied natural commensal-origin DNA, polarized HT-29 and T84 cells enhanced expression of TLR9 in a specific manner, which was subsequently associated with attenuation of TNF-alpha-induced NF-kappaB activation and NF-kappaB-mediated IL-8 expression. TLR9 silencing abolished this inhibitory effect. Apically applied LGG DNA attenuated TNF-alpha-enhanced NF-kappaB activity by reducing IkappaBalpha degradation and p38 phosphorylation. LGG DNA did not decrease the TER but rather diminished the TNF-alpha-induced TER reduction. Translocation of natural commensal-origin DNA into basolateral compartments did not occur under tested conditions., Conclusions: Our study indicates that TLR9 signaling mediates, at least in part, the anti-inflammatory effects of natural commensal-origin DNA on the gut because TLR9 silencing abolished the inhibitory effect of natural commensal-origin DNA on TNF-alpha-induced IL-8 secretion in polarized IECs. The nature of the TLR9 agonist, the polarity of cells, and the tight junction integrity of IECs has to be taken into account in order to predict the outcome of TLR9 signaling. (Inflamm Bowel Dis 2010).

Background: We previously described a potent recombinant HIV-1 neutralizing protein, sCD4-17b, composed of soluble CD4 attached via a flexible polypeptide linker to an SCFv of the 17b human monoclonal antibody directed against the highly conserved CD4-induced bridging sheet of gp120 involved in coreceptor binding. The sCD4 moiety of the bifunctional protein binds to gp120 on free virions, thereby enabling the 17b SCFv moiety to bind and block the gp120/coreceptor interaction required for entry. The previous studies using the MAGI-CCR5 assay system indicated that sCD4-17b (in concentrated cell culture medium, or partially purified) potently neutralized several genetically diverse HIIV-1 primary isolates; however, at the concentrations tested it was ineffective against several other strains despite the conservation of binding sites for both CD4 and 17b. To address this puzzle, we designed variants of sCD4-17b with different linker lengths, and tested the neutralizing activities of the immunoaffinity purified proteins over a broader concentration range against a large number of genetically diverse HIV-1 primary isolates, using the TZM-bl Env pseudotype assay system. We also examined the sCD4-17b sensitivities of isogenic viruses generated from different producer cell types., Results: We observed that immunoaffinity purified sCD4-17b effectively neutralized HIV-1 pseudotypes, including those from HIV-1 isolates previously found to be relatively insensitive in the MAGI-CCR5 assay. The potencies were equivalent for the original construct and a variant with a longer linker, as observed with both pseudotype particles and infectious virions; by contrast, a construct with a linker too short to enable simultaneous binding of the sCD4 and 17b SCFv moieties was much less effective. sCD4-17b displayed potent neutralizing activity against 100% of nearly 4 dozen HIV-1 primary isolates from diverse genetic subtypes (clades A, B, C, D, F, and circulating recombinant forms AE and AG). The neutralization breadth and potency were superior to what have been reported for the broadly neutralizing monoclonal antibodies IgG b12, 2G12, 2F5, and 4E10. The activity of sCD4-17b was found to be similar against isogenic virus particles from infectious molecular clones derived either directly from the transfected producer cell line or after a single passage through PBMCs; this contrasted with the monoclonal antibodies, which were less potent against the PMBC-passaged viruses., Conclusions: The results highlight the extremely potent and broad neutralizing activity of sCD4-17b against genetically diverse HIV-1 primary isolates. The bifunctional protein has potential applications for antiviral approaches to combat HIV infection.

The TAR RNA of HIV was engineered as an siRNA delivery vehicle to develop a combinatorial therapeutic approach. The TAR backbone was found to be a versatile backbone for expressing siRNAs. Upon expression in human cells, pronounced and specific inhibition of reporter gene expression was observed with TARmiR. The resulting TARmiR construct retained its ability to bind Tat and mediate RNAi. TARmiR was able to inhibit HIV gene expression as a TAR decoy and by RNA interference when challenged with infectious proviral DNA. The implications of this dual function therapeutic would be discussed.

Background: Influenza is a respiratory disease that seriously threatens human health. In fact, influenza virus itself does not make critical contribution to mortality induced by influenza, but "cytokine storm" produced by the excessive immune response triggered by the virus can result in inflammatory reaction of lung tissues and fatal lung tissue injury, and thus increase influenza mortality. Therefore, besides antiviral drugs, immunosuppression drugs should also be included in infection treatment., Presentation of the Hypothesis: Complement is the center of inflammatory reaction. If complement system is over activated, the body will have strong inflammatory reaction or tissue injury, resulting in pathological process. Many studies have proved that, inflammatory injury of lung tissues caused by influenza virus is closely related to complement activation. Therefore, inhibiting complement activation can significantly reduce inflammatory injury in lung tissues. As complement is both a physiological defense and pathological damage medium, systematic inhibition may result in side effects including infection. Therefore, we design targeting complement inhibitors for complement activation sites, i.e. with CR2 as targeting vector, complement inhibitors like CD59 and Crry are targeted to inflammatory sites to specially inhibit the complement activation in local injury, thus local inflammatory reaction is inhibited., Testing the Hypothesis: CR2-CD59 and CR2-Crry targeting complement inhibitors are fusion-expressed, and their biological activity is examined via in vivo and in vitro tests. CR2 targeting complement inhibitors are used to treat mouse influenza viral pneumonia model, with PBS treatment group as the control. The survival and lung tissue injury of the mice is observed and the effect of CR2 targeting complement inhibitors on pneumonia induced by influenza virus is evaluated., Implications of the Hypothesis: CR2 targeting complement inhibitors are expected to be ideal drugs for viral pneumonia.