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.     

Biomass Yield and Greenhouse Gas Emissions from a Drained Fen Peatland Cultivated with Reed Canary Grass under Different Harvest and Fertilizer Regimes

Reed canary grass (RCG, Phalaris arundinacea L.) is a suitable energy crop for cultivation in northern peatlands. However, the atmospheric impact of RCG cultivation as influenced by harvest frequency and fertilization is not clear. Here, we compared the biomass yield and greenhouse gas (GHG) balance for RCG cultivation in peatlands affected by cutting frequency and fertilizer managements. The managements included one-cut (OC) and two-cut (TC) systems that were either fertilized (TC-F) or unfertilized (TC-U) after the first cut in summer. Biomass yield of OC, TC-F and TC-U were 12, 16 and 11 Mg dry biomass per hectare per year, respectively. GHG fluxes of CO₂, N₂O and CH₄ were measured with closed chamber techniques in the period between first and second (final) harvest of the TC managements, i.e. from 15 June to 23 September 2011. In the GHG monitoring period of 100 days, all systems were net sources of CO₂ corresponding to 64?±?3, 217?±?15 and 50?±?23 g?CO₂-C?m^?2 (mean?±?standard error, n?=?3) from the OC, TC-F and TC-U systems, respectively. In the same period, emissions of N₂O from TC-F were ten times higher as compared to OC and TC-U. Emissions of CH₄ were negligible from all systems. The TC systems could not improve the GHG balance during cultivation (271, 663 and 210 g CO₂e-C?m^?2 emissions from the OC, TC-F and TC-U systems, respectively), but in a broader GHG life cycle perspective, the increased biomass yield by TC-F could replace more fossil fuel and offset at least some of the higher emissions from the system.     

The study of genetic diversity patterns of Coffea commersoniana, an endangered coffee species from Madagascar: a model for conservation of other littoral forest species

Madagascar has 59 described species of Coffea, of which 42 are listed as critically endangered, endangered, or vulnerable by the criteria of the Red List Category system of the World Conservation Union (IUCN). The littoral forest of Madagascar is a distinctive type of humid evergreen forest restricted to unconsolidated sand located within a few kilometers of the Indian Ocean, now persisting only as small fragments with ca. 10 % of its original range remaining. In an attempt to understand the genetic diversity of Madagascan coffee species, we studied ex situ and in situ populations of Coffea commersoniana, an endemic species of the littoral forests of southeastern Madagascar and soon to be impacted by mining activities in that region. The in situ populations studied showed higher genetic diversity than the ex situ population. The genetic partitioning among the two in situ populations of C. commersoniana was high enough to necessitate keeping the two populations separate for restoration purposes. Based on these findings, recommendations for conservation management (in situ and ex situ) are made.     

Improved Detection of Remote Homologues Using Cascade PSI-BLAST: Influence of Neighbouring Protein Families on Sequence Coverage

Background

Development of sensitive sequence search procedures for the detection of distant relationships between proteins at superfamily/fold level is still a big challenge. The intermediate sequence search approach is the most frequently employed manner of identifying remote homologues effectively. In this study, examination of serine proteases of prolyl oligopeptidase, rhomboid and subtilisin protein families were carried out using plant serine proteases as queries from two genomes including A. thaliana and O. sativa and 13 other families of unrelated folds to identify the distant homologues which could not be obtained using PSI-BLAST.

Methodology/Principal Findings

We have proposed to start with multiple queries of classical serine protease members to identify remote homologues in families, using a rigorous approach like Cascade PSI-BLAST. We found that classical sequence based approaches, like PSI-BLAST, showed very low sequence coverage in identifying plant serine proteases. The algorithm was applied on enriched sequence database of homologous domains and we obtained overall average coverage of 88% at family, 77% at superfamily or fold level along with specificity of ∼100% and Mathew’s correlation coefficient of 0.91. Similar approach was also implemented on 13 other protein families representing every structural class in SCOP database. Further investigation with statistical tests, like jackknifing, helped us to better understand the influence of neighbouring protein families.

Conclusions/Significance

Our study suggests that employment of multiple queries of a family for the Cascade PSI-BLAST searches is useful for predicting distant relationships effectively even at superfamily level. We have proposed a generalized strategy to cover all the distant members of a particular family using multiple query sequences. Our findings reveal that prior selection of sequences as query and the presence of neighbouring families can be important for covering the search space effectively in minimal computational time. This study also provides an understanding of the ‘bridging’ role of related families.     

A Glycolipid Adjuvant, 7DW8-5, Enhances CD8+ T Cell Responses Induced by an Adenovirus-Vectored Malaria Vaccine in Non-Human Primates

A key strategy to a successful vaccine against malaria is to identify and develop new adjuvants that can enhance T-cell responses and improve protective immunity. Upon co-administration with a rodent malaria vaccine in mice, 7DW8-5, a recently identified novel analog of α-galactosylceramide (α-GalCer), enhances the level of malaria-specific protective immune responses more strongly than the parent compound. In this study, we sought to determine whether 7DW8-5 could provide a similar potent adjuvant effect on a candidate human malaria vaccine in the more relevant non-human primate (NHP) model, prior to committing to clinical development. The candidate human malaria vaccine, AdPfCA (NMRC-M3V-Ad-PfCA), consists of two non-replicating recombinant adenoviral (Ad) vectors, one expressing the circumsporozoite protein (CSP) and another expressing the apical membrane antigen-1 (AMA1) of Plasmodium falciparum. In several phase 1 clinical trials, AdPfCA was well tolerated and demonstrated immunogenicity for both humoral and cell-mediated responses. In the study described herein, 25 rhesus macaques received prime and boost intramuscular (IM) immunizations of AdPfCA alone or with an ascending dose of 7DW8-5. Our results indicate that 7DW8-5 is safe and well-tolerated and provides a significant enhancement (up to 9-fold) in malaria-specific CD8+ T-cell responses after both priming and boosting phases, supporting further clinical development.     

Trends in inulinase production – a review

This article highlights the research work carried out in the production of inulinases from various inulin substrates using strains of bacteria, yeast and fungi. Inulin is one of the numerous polysaccharides of plant origin that contains glucose or fructose. It is used as a substrate in industrial fermentation processes and in food industries due to its relatively cheap and abundant source for the microbiological production of high-fructose syrups, ethanol and acetone–butanol. The various oligosaccharides derived from inulin also find their application in the medical and dietary sector. The inulinase acts on the β-(2,1)-D-fructoside links in inulin releasing D-fructose. Hence, this article illustrates the capability of various microbes in hydrolyzing the carbon at its optimum nutrient concentration and operating condition towards inulinase production.     

Structural and Biochemical Characterization of Compounds Inhibiting Mycobacterium tuberculosis Pantothenate Kinase

Mycobacterium tuberculosis, the bacterial causative agent of tuberculosis, currently affects millions of people. The emergence of drug-resistant strains makes development of new antibiotics targeting the bacterium a global health priority. Pantothenate kinase, a key enzyme in the universal biosynthesis of the essential cofactor CoA, was targeted in this study to find new tuberculosis drugs. The biochemical characterizations of two new classes of compounds that inhibit pantothenate kinase from M. tuberculosis are described, along with crystal structures of their enzyme-inhibitor complexes. These represent the first crystal structures of this enzyme with engineered inhibitors. Both classes of compounds bind in the active site of the enzyme, overlapping with the binding sites of the natural substrate and product, pantothenate and phosphopantothenate, respectively. One class of compounds also interferes with binding of the cofactor ATP. The complexes were crystallized in two crystal forms, one of which is in a new space group for this enzyme and diffracts to the highest resolution reported for any pantothenate kinase structure. These two crystal forms allowed, for the first time, modeling of the cofactor-binding loop in both open and closed conformations. The structures also show a binding mode of ATP different from that previously reported for the M. tuberculosis enzyme but similar to that in the pantothenate kinases of other organisms.