A Single Intramuscular Vaccination of Mice with the HSV-1 VC2 Virus with Mutations in the Glycoprotein K and the Membrane Protein UL20 Confers Full Protection against Lethal Intravaginal Challenge with Virulent HSV-1 and HSV-2 Strains

Herpes Simplex Virus type-1 (HSV-1) and type-2 (HSV-2) establish life-long infections and cause significant orofacial and genital infections in humans. HSV-1 is the leading cause of infectious blindness in the western world. Currently, there are no available vaccines to protect against herpes simplex infections. Recently, we showed that a single intramuscular immunization with an HSV-1(F) mutant virus lacking expression of the viral glycoprotein K (gK), which prevents the virus from entering into distal axons of ganglionic neurons, conferred significant protection against either virulent HSV-1(McKrae) or HSV-2(G) intravaginal challenge in mice. Specifically, 90% of the mice were protected against HSV-1(McKrae) challenge, while 70% of the mice were protected against HSV-2(G) challenge. We constructed the recombinant virus VC2 that contains specific mutations in gK and the membrane protein UL20 preventing virus entry into axonal compartments of neurons, while allowing efficient replication in cell culture, unlike the gK-null virus, which has a major defect in virus replication and spread. Intramuscular injection of mice with 10⁷ VC2 plaque forming units did not cause any significant clinical disease in mice. A single intramuscular immunization with the VC2 virus protected 100% of mice against lethal intravaginal challenge with either HSV-1(McKrae) or HSV-2(G) viruses. Importantly, vaccination with VC2 produced robust cross protective humoral and cellular immunity that fully protected vaccinated mice against lethal disease. Quantitative PCR did not detect any viral DNA in ganglionic tissues of vaccinated mice, while unvaccinated mice contained high levels of viral DNA. The VC2 virus may serve as an efficient vaccine against both HSV-1 and HSV-2 infections, as well as a safe vector for the production of vaccines against other viral and bacterial pathogens.     

Echicetin Coated Polystyrene Beads: A Novel Tool to Investigate GPIb-Specific Platelet Activation and Aggregation

von Willebrand factor/ristocetin (vWF/R) induces GPIb-dependent platelet agglutination and activation of αIIbβ3 integrin, which also binds vWF. These conditions make it difficult to investigate GPIb-specific signaling pathways in washed platelets. Here, we investigated the specific mechanisms of GPIb signaling using echicetin-coated polystyrene beads, which specifically activate GPIb. We compared platelet activation induced by echicetin beads to vWF/R. Human platelets were stimulated with polystyrene beads coated with increasing amounts of echicetin and platelet activation by echicetin beads was then investigated to reveal GPIb specific signaling. Echicetin beads induced αIIbβ3-dependent aggregation of washed platelets, while under the same conditions vWF/R treatment led only to αIIbβ3-independent platelet agglutination. The average distance between the echicetin molecules on the polystyrene beads must be less than 7 nm for full platelet activation, while the total amount of echicetin used for activation is not critical. Echicetin beads induced strong phosphorylation of several proteins including p38, ERK and PKB. Synergistic signaling via P2Y₁₂ and thromboxane receptor through secreted ADP and TxA₂, respectively, were important for echicetin bead triggered platelet activation. Activation of PKG by the NO/sGC/cGMP pathway inhibited echicetin bead-induced platelet aggregation. Echicetin-coated beads are powerful and reliable tools to study signaling in human platelets activated solely via GPIb and GPIb-triggered pathways.     

EpCAM Knockdown Alters MicroRNA Expression in Retinoblastoma- Functional Implication of EpCAM Regulated MiRNA in Tumor Progression

The co-ordinated regulation of oncogenes along with miRNAs play crucial role in carcinogenesis. In retinoblastoma (RB), several miRNAs are known to be differentially expressed. Epithelial cell adhesion molecule (EpCAM) gene is involved in many epithelial cancers including, retinoblastoma (RB) tumorigenesis. EpCAM silencing effectively reduces the oncogenic miR-17-92 cluster. In order to investigate whether EpCAM has wider effect as an inducer or silencer of miRNAs, we performed a global microRNA expression profile in EpCAM siRNA knockdown Y79 cells. MicroRNA profiling in EpCAM silenced Y79 cells showed seventy-three significantly up regulated and thirty-six down regulated miRNAs. A subset of these miRNAs was also validated in tumors. Functional studies on Y79 and WERI-Rb-1 cells transfected with antagomirs against two miRNAs of miR-181c and miR-130b showed striking changes in tumor cell properties in RB cells. Treatment with anti-miR-181c and miR-130b showed significant decrease in cell viability and cell invasion. Increase in caspase-3 level was noticed in antagomir transfected cell lines indicating the induction of apoptosis. Possible genes altered by EpCAM influenced microRNAs were predicted by bioinformatic tools. Many of these belong to pathways implicated in cancer. The study shows significant influence of EpCAM on global microRNA expression. EpCAM regulated miR-181c and miR-130b may play significant roles in RB progression. EpCAM based targeted therapies may reduce carcinogenesis through several miRNAs and target genes.     

Enhanced Production of Chikungunya Virus-Like Particles Using a High-pH Adapted Spodoptera frugiperda Insect Cell Line

Chikungunya virus-like particles (VLPs) have potential to be used as a prophylactic vaccine based on testing in multiple animal models and are currently being evaluated for human use in a Phase I clinical trial. The current method for producing these enveloped alphavirus VLPs by transient gene expression in mammalian cells presents challenges for scalable and robust industrial manufacturing, so the insect cell baculovirus expression vector system was evaluated as an alternative expression technology. Subsequent to recombinant baculovirus infection of Sf21 cells in standard culture media (pH 6.2–6.4), properly processed Chikungunya structural proteins were detected and assembled capsids were observed. However, an increase in culture pH to 6.6–6.8 was necessary to produce detectable concentrations of assembled VLPs. Since this elevated production pH exceeds the optimum for growth medium stability and Sf21 culture, medium modifications were made and a novel insect cell variant (SfBasic) was derived by exposure of Sf21 to elevated culture pH for a prolonged period of time. The high-pH adapted SfBasic insect cell line described herein is capable of maintaining normal cell growth into the typical mammalian cell culture pH range of 7.0–7.2 and produces 11-fold higher Chikungunya VLP yields relative to the parental Sf21 cell line. After scale-up into stirred tank bioreactors, SfBasic derived VLPs were chromatographically purified and shown to be similar in size and structure to a VLP standard derived from transient gene expression in HEK293 cells. Total serum anti-Chikungunya IgG and neutralizing titers from guinea pigs vaccinated with SfBasic derived VLPs or HEK293 derived VLPs were not significantly different with respect to production method, suggesting that this adapted insect cell line and production process could be useful for manufacturing Chikungunya VLPs for use as a vaccine. The adaptation of Sf21 to produce high levels of recombinant protein and VLPs in an elevated pH range may also have applications for other pH-sensitive protein or VLP targets.     

Studies in the secretory glands of Hiptage sericea (Malpighiaceae)

Hiptage sericea is shown to possess both lipophilic glands and extrafloral nectaries. Both types of glands develop from a group of initials and show similarities in organisation of tissue systems and secretion. The nature of the secretory substances is however different. The occurrence and function of the glands are discussed.     

Rain forest expansion mediated by successional processes in vegetation thickets in the Western Ghats of India

Aim The objective of this study was to document succession from grassland thickets to rain forest, and to provide evidence for their potential as restoration tools. Location The Linganamakki region (State of Karnataka) of the Central Western Ghats of India. Method We selected thirty vegetation thickets ranging from 4 to 439 m² in area in the vicinity of rain forest. The area of each small thicket was estimated as an oval using its maximum length and its maximum width. When the shape was irregular (mostly in large thickets) the limits of the thicket were mapped and the area calculated from the map. Plant species were identified, the number of individuals was estimated and their heights measured. Results There was a progression in the thickets from early to late successional species. Small thickets were characterized by ecotone species and savanna trees such as Catunaregam dumetorum. Savanna trees served as a nucleus for thicket formation. Colonizing species were mostly bird‐dispersed. As succession proceeded in larger thickets, the proportion of evergreen, late‐successional rain forest trees increased. The species composition of the large thickets differed depending on the species composition of reproductive adults in the nearby forested areas. The species within small thickets were also found in the large thickets. The nestedness in species composition suggested that species turnover was deterministic based on thicket size. Human disturbance (leaf and wood collection by the local populations) affected the species composition and the species–area relationship of thickets. Main conclusions Vegetation thickets are nodal centres for rain forest colonization within grasslands. They expand and replace savanna. Early successional bird‐dispersed species established around savanna trees followed by late‐successional rain forest trees dispersed from the nearby forest by birds. Restoration programmes that reproduce natural successional processes such as those observed in thickets will be more successful and less expensive than the methods currently being employed, where trees are individually planted in grassland. Wood harvesting is the only factor that prevents thicket growth and coalescence and hampers forest expansion.