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.     

Japanese Encephalitis Virus Disrupts Cell-Cell Junctions and Affects the Epithelial Permeability Barrier Functions

Japanese encephalitis virus (JEV) is a neurotropic flavivirus, which causes viral encephalitis leading to death in about 20–30% of severely-infected people. Although JEV is known to be a neurotropic virus its replication in non-neuronal cells in peripheral tissues is likely to play a key role in viral dissemination and pathogenesis. We have investigated the effect of JEV infection on cellular junctions in a number of non-neuronal cells. We show that JEV affects the permeability barrier functions in polarized epithelial cells at later stages of infection. The levels of some of the tight and adherens junction proteins were reduced in epithelial and endothelial cells and also in hepatocytes. Despite the induction of antiviral response, barrier disruption was not mediated by secreted factors from the infected cells. Localization of tight junction protein claudin-1 was severely perturbed in JEV-infected cells and claudin-1 partially colocalized with JEV in intracellular compartments and targeted for lysosomal degradation. Expression of JEV-capsid alone significantly affected the permeability barrier functions in these cells. Our results suggest that JEV infection modulates cellular junctions in non-neuronal cells and compromises the permeability barrier of epithelial and endothelial cells which may play a role in viral dissemination in peripheral tissues.     

Mutations in a guanylate cyclase GCY-35/GCY-36 modify Bardet-Biedl syndrome-associated phenotypes in Caenorhabditis elegans

Ciliopathies are pleiotropic and genetically heterogeneous disorders caused by defective development and function of the primary cilium. Bardet-Biedl syndrome (BBS) proteins localize to the base of cilia and undergo intraflagellar transport, and the loss of their functions leads to a multisystemic ciliopathy. Here we report the identification of mutations in guanylate cyclases (GCYs) as modifiers of Caenorhabditis elegans bbs endophenotypes. The loss of GCY-35 or GCY-36 results in suppression of the small body size, developmental delay, and exploration defects exhibited by multiple bbs mutants. Moreover, an effector of cGMP signalling, a cGMP-dependent protein kinase, EGL-4, also modifies bbs mutant defects. We propose that a misregulation of cGMP signalling, which underlies developmental and some behavioural defects of C. elegans bbs mutants, may also contribute to some BBS features in other organisms.     

Disheveled mediated planar cell polarity signaling is required in the second heart field lineage for outflow tract morphogenesis

Disheveled (Dvl) is a key regulator of both the canonical Wnt and the planar cell polarity (PCP) pathway. Previous genetic studies in mice indicated that outflow tract (OFT) formation requires Dvl1 and 2, but it was unclear which pathway was involved and whether Dvl1/2-mediated signaling was required in the second heart field (SHF) or the cardiac neural crest (CNC) lineage, both of which are critical for OFT development. In this study, we used Dvl1/2 null mice and a set of Dvl2 BAC transgenes that function in a pathway-specific fashion to demonstrate that Dvl1/2-mediated PCP signaling is essential for OFT formation. Lineage-specific gene-ablation further indicated that Dvl1/2 function is dispensable in the CNC, but required in the SHF for OFT lengthening to promote cardiac looping. Mutating the core PCP gene Vangl2 and non-canonical Wnt gene Wnt5a recapitulated the OFT morphogenesis defects observed in Dvl1/2 mutants. Consistent with genetic interaction studies suggesting that Wnt5a signals through the PCP pathway, Dvl1/2 and Wnt5a mutants display aberrant cell packing and defective actin polymerization and filopodia formation specifically in SHF cells in the caudal splanchnic mesoderm (SpM), where Wnt5a and Dvl2 are co-expressed specifically. Our results reveal a critical role of PCP signaling in the SHF during early OFT lengthening and cardiac looping and suggest that a Wnt5a→ Dvl PCP signaling cascade may regulate actin polymerization and protrusive cell behavior in the caudal SpM to promote SHF deployment, OFT lengthening and cardiac looping.     

Capturing the Dynamics of a Hybrid Multiscale Cancer Model with a Continuum Model

Cancer is a complex disease involving processes at spatial scales from subcellular, like cell signalling, to tissue scale, such as vascular network formation. A number of multiscale models have been developed to study the dynamics that emerge from the coupling between the intracellular, cellular and tissue scales. Here, we develop a continuum partial differential equation model to capture the dynamics of a particular multiscale model (a hybrid cellular automaton with discrete cells, diffusible factors and an explicit vascular network). The purpose is to test under which circumstances such a continuum model gives equivalent predictions to the original multiscale model, in the knowledge that the system details are known, and differences in model results can be explained in terms of model features (rather than unknown experimental confounding factors). The continuum model qualitatively replicates the dynamics from the multiscale model, with certain discrepancies observed owing to the differences in the modelling of certain processes. The continuum model admits travelling wave solutions for normal tissue growth and tumour invasion, with similar behaviour observed in the multiscale model. However, the continuum model enables us to analyse the spatially homogeneous steady states of the system, and hence to analyse these waves in more detail. We show that the tumour microenvironmental effects from the multiscale model mean that tumour invasion exhibits a so-called pushed wave when the carrying capacity for tumour cell proliferation is less than the total cell density at the tumour wave front. These pushed waves of tumour invasion propagate by triggering apoptosis of normal cells at the wave front. Otherwise, numerical evidence suggests that the wave speed can be predicted from linear analysis about the normal tissue steady state.     

Silver nitrate-induced in vitro shoot multiplication and precocious flowering in <Emphasis Type="Italic">Catharanthus roseus</Emphasis> (L.) G. Don,a rich source of terpenoid indole alkaloids

Catharanthus roseus (L.) G. Don is an economically and medicinally important plant since its leaves and flowers contain terpenoid indole alkaloids. The present study, for the first time, encompasses the influence of silver nitrate (AgNO₃), in consort with cytokinins like N ⁶-benzyladenine (BA) and 6-furfurylaminopurine (kinetin), to regenerate multiple shoots from nodal segments explants and to induce high-frequency precocious flowering of C. roseus under in vitro condition. Synergistic effect of equal concentrations of BA and kinetin was enhanced following the amalgamation of AgNO₃. As high as 98% explants responded to multiple shoot initiation and proliferation in Murashige and Skoog medium supplemented with 3 µM BA, 3 µM kinetin and 0.1 µM AgNO₃. As many as 7 shoots were developed per explant following 12 days of inoculation. Continuous culture in the same medium for 21 days induced precocious flowering from 75% shoots, wherein a maximum of ~?6 (5.67?±?0.88) flowers was observed per in vitro shoot. On the other hand, in the combinations of BA and kinetin excluding AgNO₃, a maximum of 6.67% explants responded and initiated merely 3.33 shoots per explant. Nevertheless, no induction of flower was observed in the media devoid of AgNO₃. Our results on the induction and proliferation of multiple shoots with simultaneous flowering would help the global pharmaceutical industry to produce in vitro shoots and flowers in bulk, as an alternative source of alkaloids.     

Role of cervical dendritic cell subsets,co-stimulatory molecules,cytokine secretion profile and beta-estradiol in development of sequalae to Chlamydia trachomatis infection

Background  

Chlamydia trachomatis infection of the female genital tract can lead to serious sequelae resulting in fertility related disorders. Little is known about the mechanism leading to Chlamydia induced pathology and factors responsible for it. As only some of the women develops reproductive disorders while majority of the women clears infection without any severe sequalae, mucosal immune response in women with or without fertility disorders was studied to identify factors which may lead to final clinical outcome of chlamydial infection.     

Virus antibody dynamics in primary and secondary dengue infections

Dengue viral infections show unique infection patterns arising from its four serotypes, (DENV-1,2,3,4). Its effects range from simple fever in primary infections to potentially fatal secondary infections. We analytically and numerically analyse virus dynamics and humoral response in a host during primary and secondary dengue infection for long periods using micro-epidemic models. The models presented here incorporate time delays, antibody dependent enhancement, a dynamic switch and a correlation factor between different DENV serotypes. We find that the viral load goes down to undetectable levels within 7–14 days as is observed for dengue infection, in both cases. For primary infection, the stability analysis of steady states shows interesting dependence on the time delay involved in the production of antibodies from plasma cells. We demonstrate the existence of a critical value for the immune response parameter, beyond which the infection gets completely cured. For secondary infections with a different serotype, the homologous antibody production is enhanced due to the influence of heterologous antibodies. The antibody production is also controlled by the correlation factor, which is a measure of similarities between the different DENV serotypes involved. Our results agree with clinically observed humoral responses for primary and secondary infections.