Synthesis and Properties of 2′-O-Methylribonucleotide Methylphosphonate Containing Chimeric Oligonucleotides

Abstract

2′-O-methylribonucleoside methylphosphonamidites are synthesized and incorporated into oligonucleotides to obtain chimeric antisense oligonucleotides. The resulting oligonucleotide binds to their target RNA/DNA sequences efficiently and stable in a medium containing bovine serum.     

Destabilization of DNA Guanine Quadruplex Structure by Foldback Triplex-Forming Oligodeoxynucleotides

Abstract

An oligodeoxynucleotide designed to bind to a single stranded guanine rich DNA sequence through Watson-Crick followed by Hoogsteen hydrogen bonds was found to destabilize quadruplex structure formed by the target sequence. However, the conventional antisense and antigene oligonucleotides were unable to destabilize the same quadruplex structure.     

Oligonucleotides Containing Acyclic Nucleoside Analogues with Carbamate Internucleoside Linkages

Abstract

Synthesis of 2′-deoxy-2′,3′-secothymidine t and its dimer t?t, where the two 2′-deoxy-2′,3′-secothymidine t units are connected via a carbamate, ?=3′-NH-CO-O-5′, internucleoside linkage has been achieved. These building blocks were protected in the 5′-position, converted into their phosphoramidites, or attached onto CPG, and then used for “chimeric oligonucleotide” synthesis.     

Mixed-Backbone Oligonucleotides Containing Phosphorothioate and Methylphosphonate Linkages as Second Generation Antisense Oligonucleotide

Abstract

Antisense oligonucleotides are being studied as novel therapeutic agents. To further improve the properties of antisense oligonucleotides, we have synthesized phosphorothioate oligonucleotides containing methylphosphonate linkages at the 5′-end, the 3′-end, or in the center, and have evaluated the impact of these linkages on the biophysical properties, biological properties, and some of the safety parameters.     

Tandem combination of Trigonella foenum-graecum defensin (Tfgd2) and Raphanus sativus antifungal protein (RsAFP2) generates a more potent antifungal protein

Plant defensins are small (45 to 54 amino acids) positively charged antimicrobial peptides produced by the plant species, which can inhibit the growth of a broad range of fungi at micro-molar concentrations. These basic peptides share a common characteristic three-dimensional folding pattern with one α-helix and three β-sheets that are stabilized by eight disulfide-linked cysteine residues. Instead of using two single-gene constructs, it is beneficial when two effective genes are made into a single fusion gene with one promoter and terminator. In this approach, we have linked two plant defensins namely Trigonella foenum-graecum defensin 2 (Tfgd2) and Raphanus sativus antifungal protein 2 (RsAFP2) genes by a linker peptide sequence (occurring in the seeds of Impatiens balsamina) and made into a single-fusion gene construct. We used pET-32a+ vector system to express Tfgd2-RsAFP2 fusion gene with hexahistidine tag in Escherichia coli BL21 (DE3) pLysS cells. Induction of these cells with 1 mM IPTG achieved expression of the fusion protein. The solubilized His₆-tagged recombinant fusion protein was purified by immobilized-metal (Ni²⁺) affinity column chromatography. The final yield of the fusion protein was 500 ng/μL. This method produced biologically active recombinant His₆-tagged fusion protein, which exhibited potent antifungal action towards the plant pathogenic fungi (Botrytis cinerea, Fusarium moniliforme, Fusarium oxysporum, Phaeoisariopsis personata and Rhizoctonia solani along with an oomycete pathogen Phytophthora parasitica var nicotianae) at lower concentrations under in vitro conditions. This strategy of combining activity of two defensin genes into a single-fusion gene will definitely be a promising utility for biotechnological applications.     

Bispidine-Amino Acid Conjugates Act as a Novel Scaffold for the Design of Antivirals That Block Japanese Encephalitis Virus Replication

Background

Japanese encephalitis virus (JEV) is a major cause of viral encephalitis in South and South-East Asia. Lack of antivirals and non-availability of affordable vaccines in these endemic areas are a major setback in combating JEV and other closely related viruses such as West Nile virus and dengue virus. Protein secondary structure mimetics are excellent candidates for inhibiting the protein-protein interactions and therefore serve as an attractive tool in drug development. We synthesized derivatives containing the backbone of naturally occurring lupin alkaloid, sparteine, which act as protein secondary structure mimetics and show that these compounds exhibit antiviral properties.

Methodology/Principal Findings

In this study we have identified 3,7-diazabicyclo[3.3.1]nonane, commonly called bispidine, as a privileged scaffold to synthesize effective antiviral agents. We have synthesized derivatives of bispidine conjugated with amino acids and found that hydrophobic amino acid residues showed antiviral properties against JEV. We identified a tryptophan derivative, Bisp-W, which at 5 µM concentration inhibited JEV infection in neuroblastoma cells by more than 100-fold. Viral inhibition was at a stage post-entry and prior to viral protein translation possibly at viral RNA replication. We show that similar concentration of Bisp-W was capable of inhibiting viral infection of two other encephalitic viruses namely, West Nile virus and Chandipura virus.

Conclusions/Significance

We have demonstrated that the amino-acid conjugates of 3,7-diazabicyclo[3.3.1]nonane can serve as a molecular scaffold for development of potent antivirals against encephalitic viruses. Our findings will provide a novel platform to develop effective inhibitors of JEV and perhaps other RNA viruses causing encephalitis.     

INPPO Actions and Recognition as a Driving Force for Progress in Plant Proteomics: Change of Guard,INPPO Update,and Upcoming Activities

The International Plant Proteomics Organization (INPPO) is a non‐profit organization whose members are scientists involved or interested in plant proteomics. Since the publication of the first INPPO highlights in 2012, continued progress on many of the organization's mandates/goals has been achieved. Two major events are emphasized in this second INPPO highlights. First, the change of guard at the top, passing of the baton from Dominique Job, INPPO founding President to Ganesh Kumar Agrawal as the incoming President. Ganesh K. Agrawal, along with Dominique Job and Randeep Rakwal initiated the INPPO. Second, the most recent INPPO achievements and future targets, mainly the organization of first the INPPO World Congress in 2014, tentatively planned for Hamburg (Germany), are mentioned.     

In vitro selection and field responses of somaclonal variant plants of rice cv PR113 for drought tolerance

Drought is the major environmental stress that limits rice productivity worldwide. In vitro somaclonal variation using different selection agents has been used for crop improvement. Here, rice plants of cv PR113 were selected in vitro on 30, 50 and 70 g L^-1 polyethylene glycol 6,000 (PEG). Callus growth, proliferation, calli volume (first and second culture) and plantlet regeneration (third culture) were found to be decreased upto a certain level to acquire tolerance to PEG-induced drought. From the field data, 30 g L^-1 PEG lines showed higher vegetative growth (plant height, tiller number, leaf number, shoot weight and root growth) as compared with 50 g L^-1 PEG selected somaclone lines under limited irrigation. The yield parameters-panicle length, panicle weight, grains per panicle, 1,000-grain weight, grain yield per plant, harvest index and grain straw ratio were also higher in 30 g L^-1 PEG lines as compared with 50 g L^-1 PEG lines. The results, therefore indicate that 30 g L^-1 PEG selected somaclone lines were more suited than 50 g L^-1 PEG selected somaclone lines under stress as compared with WT. The finding suggests that rice cv PR113 somaclones generated on PEG are found to be drought tolerant under field condition with better yield.     

Associations between Active Travel to Work and Overweight,Hypertension, and Diabetes in India: A Cross-Sectional Study

Background

Increasing active travel (walking, bicycling, and public transport) is promoted as a key strategy to increase physical activity and reduce the growing burden of noncommunicable diseases (NCDs) globally. Little is known about patterns of active travel or associated cardiovascular health benefits in low- and middle-income countries. This study examines mode and duration of travel to work in rural and urban India and associations between active travel and overweight, hypertension, and diabetes.

Methods and Findings

Cross-sectional study of 3,902 participants (1,366 rural, 2,536 urban) in the Indian Migration Study. Associations between mode and duration of active travel and cardiovascular risk factors were assessed using random-effect logistic regression models adjusting for age, sex, caste, standard of living, occupation, factory location, leisure time physical activity, daily fat intake, smoking status, and alcohol use. Rural dwellers were significantly more likely to bicycle (68.3% versus 15.9%; p<0.001) to work than urban dwellers. The prevalence of overweight or obesity was 50.0%, 37.6%, 24.2%, 24.9%; hypertension was 17.7%, 11.8%, 6.5%, 9.8%; and diabetes was 10.8%, 7.4%, 3.8%, 7.3% in participants who travelled to work by private transport, public transport, bicycling, and walking, respectively. In the adjusted analysis, those walking (adjusted risk ratio [ARR] 0.72; 95% CI 0.58–0.88) or bicycling to work (ARR 0.66; 95% CI 0.55–0.77) were significantly less likely to be overweight or obese than those travelling by private transport. Those bicycling to work were significantly less likely to have hypertension (ARR 0.51; 95% CI 0.36–0.71) or diabetes (ARR 0.65; 95% CI 0.44–0.95). There was evidence of a dose-response relationship between duration of bicycling to work and being overweight, having hypertension or diabetes. The main limitation of the study is the cross-sectional design, which limits causal inference for the associations found.

Conclusions

Walking and bicycling to work was associated with reduced cardiovascular risk in the Indian population. Efforts to increase active travel in urban areas and halt declines in rural areas should be integral to strategies to maintain healthy weight and prevent NCDs in India. Please see later in the article for the Editors'' Summary