Geraniin extracted from the rind of <Emphasis Type="Italic">Nephelium lappaceum</Emphasis> binds to dengue virus type-2 envelope protein and inhibits early stage of virus replication

Background

The rapid rise and spread in dengue cases, together with the unavailability of safe vaccines and effective antiviral drugs, warrant the need to discover and develop novel anti-dengue treatments. In this study the antiviral activity of geraniin, extracted from the rind of Nephelium lappaceum, against dengue virus type-2 (DENV-2) was investigated.

Methods

Geraniin was prepared from Nephelium lappaceum rind by reverse phase C-18 column chromatography. Cytotoxicity of geraniin towards Vero cells was evaluated using MTT assay while IC₅₀ value was determined by plaque reduction assay. The mode-of-action of geraniin was characterized using the virucidal, attachment, penetration and the time-of-addition assays’. Docking experiments with geraniin molecule and the DENV envelope (E) protein was also performed. Finally, recombinant E Domain III (rE-DIII) protein was produced to physiologically test the binding of geraniin to DENV-2 E-DIII protein, through ELISA competitive binding assay.

Results

Cytotoxicity assay confirmed that geraniin was not toxic to Vero cells, even at the highest concentration tested. The compound exhibited DENV-2 plaque formation inhibition, with an IC₅₀ of 1.75 μM. We further revealed that geraniin reduced viral infectivity and inhibited DENV-2 from attaching to the cells but had little effect on its penetration. Geraniin was observed to be most effective when added at the early stage of DENV-2 infection. Docking experiments showed that geraniin binds to DENV E protein, specifically at the DIII region, while the ELISA competitive binding assay confirmed geraniin’s interaction with rE-DIII with high affinity.

Conclusions

Geraniin from the rind of Nephelium lappaceum has antiviral activity against DENV-2. It is postulated that the compound inhibits viral attachment by binding to the E-DIII protein and interferes with the initial cell-virus interaction. Our results demonstrate that geraniin has the potential to be developed into an effective antiviral treatment, particularly for early phase dengue viral infection.

     

Metabolite profiling of ascidian Styela plicata using LC–MS with multivariate statistical analysis and their antitumor activity

To identify the metabolite distribution in ascidian, we have applied an integrated liquid chromatography– tandem mass spectrometry (LC–MS) metabolomics approach to explore and identify patterns in chemical diversity of invasive ascidian Styela plicata. A total of 71 metabolites were reported among these alkaloids, fatty acids and lipids are the most dominant chemical group. Multivariate statistical analysis, principal component analysis (PCA) showed a clear separation according to chemical diversity and taxonomic groups. PCA and partial least square discriminant analysis were applied to discriminate the chemical group of S. plicata crude compounds and classify the compounds with unknown biological activities. In this study, we reported for the first time that a partially purified methanol extract prepared from the ascidian S. plicata and Ascidia mentula possess antitumor activity against four tumor cell lines with different tumor histotype, such as HeLa (cervical carcinoma), HT29 (colon carcinoma), MCF-7 (breast carcinoma) and M14 (melanoma). S. plicata fraction SP-50 showed strong inhibition of cell proliferation and induced apoptosis in HeLa and HT29 cells, thus indicating S. plicata fraction SP-50 a potential lead compound for anticancer therapy. The molecular mechanism of action and chemotherapeutic potential of these ascidian unknown biomolecules need further research.     

Eco-friendly approaches for the management of fenugreek root rot

To evolve eco-friendly management of fenugreek root rot caused by Rhizoctonia solani, a field trial was conducted during Kharif 2002 and Rabi seasons of 2002–2003 and 2003–2004. Experiments were conducted with eight treatments and three replications in RBD using the variety CO-2. The pooled analysis of the three season data showed that seed treatment with Trichoderma viride at 4g/kg of seed + soil application of Trichoderma viride at 5 kg/ha + soil application of neem cake at 150 kg/ha (T₃) recorded a percent disease index (PDI) of 23.1 versus 65.5 PDI in the control which accounted for a disease reduction of 64.7%. It was on par with seed treatment with Trichoderma viride at 4g/kg of seed + soil application of T. viride at 5 kg/ha (T₂) which reduced the disease incidence by 62.3% (24.7 PDI). The chemical treatment used for comparison, i.e. seed treatment with carbendazim + soil drenching at 0.1% + soil application of neem cake at 150 kg/ha recorded the lowest PDI of 16.8 with 74.4% disease reduction. Among the various treatments T₃ gave a seed yield of 572.7 kg/ha followed by T₂ (555.7 kg/ha). Treatment T₇ recorded the highest yield of 578.7 kg/ha. In the control plot the recorded yield was only 359.3 kg/ha. Though T₃ was more effective at reducing the disease incidence than T₂, the C:B ratio was higher (1:9.1) in respect of T₂ than T₃, which gave a C:B ratio of only 1:3.9. Hence, seed treatment with T. viride at 4g/kg + soil application of T. viride at 5kg/ha is a cost effective, eco-friendly management strategy for fenugreek root rot.     

Molecular breeding of transgenic perennial ryegrass (<Emphasis Type="Italic">Lolium perenne</Emphasis> L.) with altered fructan biosynthesis through the expression of fructosyltransferases

Perennial ryegrass is the most important forage grass used in temperate agriculture. Transgenic perennial ryegrass events with altered fructan biosynthesis have the potential not only to increase animal production by improving digestibility of the grasses, but also to increase pasture intake by the animal due to lower neutral detergent fibre (NDF) concentrations. Transgenic perennial ryegrass plants were shown to have increased (P?<?0.05) in fructan concentrations of leaf blades in transgenic T₀ events and have thus an increase in water-soluble carbohydrate and in vivo dry matter digestibility concentrations as well as a decrease in the NDF concentration within the plant in spring and summer. These changes in nutritive value have led to an increase in metabolisable energy of up to 1.7 MJ ME·kg DM^?1 in selected T₀ events compared to FLp418-20 during spring and summer, with no differences in autumn or winter. The field evaluation of these events and the further development of these events using molecular breeding technologies are described in this paper.     

Fish Heat Shock Cognate 70 Derived AMPs <Emphasis Type="Italic">Cs</Emphasis>HSC70 A1 and <Emphasis Type="Italic">Cs</Emphasis>HSC70 A2

Heat shock cognate 70 (HSC70) is an important evolutionary conserved protein that plays a major role in maintaining the homeostasis and immunity of many organisms. In this study, a HSC70 from Channa striatus was identified from its cDNA library and characterized using bioinformatics and molecular biology tools. CsHSC70 cDNA was 1953 base pair (bp) in length along with an open reading frame which encoded a polypeptide of 650 amino acid residues. Tissue distribution results showed that CsHSC70 was considerably expressed in gill, to a lesser extent in head kidney, blood, spleen and liver and at low level in other tissues. Using C. striatus gill as cell model, effects of fungal, bacterial and poly I:C stimulant on the mRNA levels of CsHSC70 was examined. We also described the antimicrobial features of two peptides namely CsHSC70 A1and CsHSC70 A2 derived from the N-terminal of CsHSC70 protein. CsHSC70 A1 peptide (40 µg/ml) exhibited potent bactericidal activity against Micrococcus luteus cells. Flow cytometric analysis revealed that the M. luteus cells stained with propidium iodide, upon treated with CsHSC70 A1 at the concentration of 40 µM/ml showed 38% survival compared to its control (99.6%). It seems that CsHSC70 A1 peptide shows antimicrobial activity against M. luteus through membrane disruption. Additionally, scanning electron microscope (SEM) observation confirmed that CsHSC70 A1 peptide treatment completely damaged and destructed the M. luteus cells. Taken together, these findings suggest that CsHSC70 A1 peptide could be a safe and potential therapeutic molecule substitute to antibiotics in various clinical fields.     

Antioxidant,antimicrobial and cytotoxicity potential of n-hexane extract of Cayratia trifolia L

It is of interest to analyze the antioxidant, antimicrobial and cytotoxicity activity of n-hexane extract of Cayratia trifolia L. (C. trifolia). The antimicrobial activity of n-hexane extract of C. trifolia was determined using disc diffusion method against six selected pathogenic microorganisms. The cytotoxicity potential of n-hexane plant extract was also studied against A2780 cell lines by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Results, n-hexane extract of C. trifolia possess significant antioxidant activity with significant IC50 values in radical scavenging assays. In antimicrobial studies, the maximum zone of inhibition was found in the range of 19.0 ± 0.1 to 22.0 ± 0.1 mm. In MTT assay, inhibition of cell growth with minimal IC50 values of 46.25±0.42μg/mL against A2780 cell lines was observed. Thus, n-hexane extract of C. trifolia is a possible antioxidant, antimicrobial and cytotoxicity agent.     

Differential proteomic profiling identifies novel molecular targets of pterostilbene against experimental diabetes

Pterostilbene (PTS), a naturally occurring stilbene, confers protection against oxidative and cytokine stress induced pancreatic β-cell apoptosis in vitro and in vivo. To provide insights into the molecular mechanism, we performed a proteomic study on the pancreas of PTS-treated diabetic mice using electrospray ionization tandem–mass spectrometry (LC–MS/MS). A total of 1,260 proteins were detected in triplicate samples. Of which, 359 proteins were found to be differentially regulated in streptozotocin-induced diabetic mice pancreas with two fold difference ( P < 0.05, two or more peptides) and on PTS treatment 315 proteins were normalized to control levels. Gene ontology (GO) indicated that majority of the differentially regulated proteins are involved in cellular functions such as metabolism, cellular structure, oxidative stress, endoplasmic-reticulum-associated protein degradation (ERAD) pathway and several stress sensors. Protein–protein interaction network analysis of these differentially expressed proteins showed clustering of proteins involved in protein processing in endoplasmic reticulum (protein synthesis machinery and protein folding), oxidative phosphorylation/oxidative stress proteins, oligosaccharide metabolic process, and antioxidant activity. Our results highlighted that PTS administration rehabilitated the defective metabolic process and redox imbalance, and also suppressed the unfolded protein response and ERAD pathways. The effects on targeting ER machinery and suppressing oxidative stress suggest the great potential of PTS for diabetes management.     

Deletion of the C-terminus of polynucleotide phosphorylase increases twitching motility, a virulence characteristic of the anaerobic bacterial pathogen Dichelobacter nodosus

The Gram-negative anaerobe Dichelobacter nodosus is the causative agent of footrot in sheep. Different strains of D. nodosus cause disease of differing severities, ranging from benign to virulent. Virulent strains have greater twitching motility and secrete proteases that are more thermostable than those secreted by benign strains. We have identified polynucleotide phosphorylase (PNPase) as a putative virulence regulator and have proposed that PNPase expression is modulated by the adjacent integration of genetic elements. In this study, we compared PNPase activity in three virulent and four benign strains of D. nodosus and found that PNPase activity is lower in virulent strains. We disrupted the pnpA gene in three benign D. nodosus strains and two virulent strains and showed that deletion of the S1 domain of PNPase reduced catalytic activity. In all but one case, deletion of the PNPase S1 domain had no effect on the thermostability of extracellular proteases. However, this deletion resulted in an increase in twitching motility in benign, but not in virulent strains. Reconstruction of the pnpA gene in two mutant benign strains reduced twitching motility to the parental level. These results support the hypothesis that PNPase is a virulence repressor in benign strains of D. nodosus .