Pathway analysis of host responses to dengue virus serotype 2 infection and inhibition of viral envelope protein by naringenin from Ganoderma lucidum.

Dengue fever cases are spiking over the last two decades. Incessant efforts are still being made to gain deeper insights on this arboviral disease and to identify bioactive antivirals. In this study, bioinformatics analysis was conducted to identify the differentially expressed genes (DEGs) in the expression profiling datasets of dengue virus serotype 2 (DENV2) patients. We found overexpressed genes in dengue patients that can interrupt cell cycle progression and phase transitions of mitosis inside the host to favour the viral replication process. These DEGs were associated with the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways such as cell cycle and DNA replication. A protein interaction network consisting of these significant pathways was also constructed using STRING. Futher, the traditional Chinese medicine (TCM) compounds from Ganoderma lucidum were screened to target DENV2 envelope protein, which was crucial for viral fusion activity. Docking, orbital energy, and toxicity prediction analysis revealed that naringenin was the best antiviral candidate. Following molecular dynamics simulations, the predicted binding energy of the protein-naringenin system using the molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) approach was slightly greater than the control system. It is recommended to perform in vitro inhibition of naringenin against DENV2 and use our findings to complement the experimental data obtained.

Scalable Production of Recombinant Membrane Active Peptides and Its Potential as a Complementary Adjunct to Conventional Chemotherapeutics.

The production of short anticancer peptides in recombinant form is an alternative method for costly chemical manufacturing. However, the limitations of host toxicity, bioactivity and column purification have impaired production in mass quantities. In this study, short cationic peptides were produced in aggregated inclusion bodies by double fusion with a central protein that has anti-cancer activity. The anticancer peptides Tachiplicin I (TACH) and Latarcin 1 (LATA) were fused with the N- and C-terminus of the MAP30 protein, respectively. We successfully produced the recombinant TACH-MAP30-LATA protein and MAP30 alone in E. coli that represented 59% and 68% of the inclusion bodies. The purified form of the inclusion bodies was prepared by eliminating host cell proteins through multiple washing steps and semi-solubilization in alkaline buffer. The purified active protein was recovered by inclusive solubilization at pH 12.5 in the presence of 2 M urea and refolded in alkaline buffer containing oxides and reduced glutathione. The peptide-fusion protein showed lower CC50 values against cancer cells (HepG2, 0.35±0.1 µM and MCF-7, 0.58±0.1 µM) compared with normal cells (WRL68, 1.83±0.2 µM and ARPE19, 2.5±0.1 µM) with outstanding activity compared with its individual components. The presence of the short peptides facilitated the entry of the peptide fusion protein into cancer cells (1.8 to 2.2-fold) compared with MAP30 alone through direct interaction with the cell membrane. The cancer chemotherapy agent doxorubicin showed higher efficiency and selectivity against cancer cells in combination with the peptide- fusion protein. This study provides new data on the mass production of short anticancer peptides as inclusion bodies in E. coli by fusion with a central protein that has similar activity. The product was biologically active against cancer cells compared with normal cells and enhanced the activity and selective delivery of an anticancer chemotherapy agent.

A combination of doxycycline and ribavirin alleviated chikungunya infection.

Lack of vaccine and effective antiviral drugs against chikungunya virus (CHIKV) outbreaks have led to significant impact on health care in the developing world. Here, we evaluated the antiviral effects of tetracycline (TETRA) derivatives and other common antiviral agents against CHIKV. Our results showed that within the TETRA derivatives group, Doxycycline (DOXY) exhibited the highest inhibitory effect against CHIKV replication in Vero cells. On the other hand, in the antiviral group Ribavirin (RIBA) showed higher inhibitory effects against CHIKV replication compared to Aciclovir (ACIC). Interestingly, RIBA inhibitory effects were also higher than all but DOXY within the TETRA derivatives group. Docking studies of DOXY to viral cysteine protease and E2 envelope protein showed non-competitive interaction with docking energy of -6.6±0.1 and -6.4±0.1 kcal/mol respectively. The 50% effective concentration (EC50) of DOXY and RIBA was determined to be 10.95±2.12 µM and 15.51±1.62 µM respectively, while DOXY+RIBA (1:1 combination) showed an EC50 of 4.52±1.42 µM. When compared, DOXY showed higher inhibition of viral infectivity and entry than RIBA. In contrast however, RIBA showed higher inhibition against viral replication in target cells compared to DOXY. Assays using mice as animal models revealed that DOXY+RIBA effectively inhibited CHIKV replication and attenuated its infectivity in vivo. Further experimental and clinical studies are warranted to investigate their potential application for clinical intervention of CHIKV disease.

Cloning, expression and characterization of the endoglucanase gene from Bacillus subtilis UMC7 isolated from the gut of the indigenous termite Macrotermes malaccensis in Escherichia coli

Background Bacillus subtilis UMC7 isolated from the gut of termite Macrotermes malaccensis has the ability to secrete a significant amount of extracellular endoglucanase, with an enzyme activity of 0.12 ± 0.01 μmol/min/mL. However, for economically viable industrial applications, the enzyme needs to be expressed in a heterologous host to overcome the low enzyme production from the wild-type strain. Results The endoglucanase gene from B. subtilis UMC7 was successfully cloned and expressed. A higher enzyme activity was observed in the intracellular fraction of the recombinant clone (0.51 ± 0.02 μmol/min/mL) compared with the cell-bound fraction (0.37 ± 0.02 μmol/min/mL) and the extracellular fraction (0.33 ± 0.01 μmol/min/mL). The recombinant endoglucanase was approximately 56 kDa, with optimal enzyme activity at 60°C and pH 6.0. The activity of the enzyme was enhanced by the addition of Ca2 +. However, the enzyme was inhibited by other metal ions in the following order: Fe3 + > Ni2 + > Cu2 + > Mn2 + = Zn2 + > Mg2 + > Cd2 + > Cr2 +. The enzyme was able to hydrolyze both low- and high-viscosity carboxymethyl-cellulose (CMC), avicel, cotton linter, filter paper and avicel but not starch, xylan, chitin, pectin and p-nitrophenyl α-d-glucopyranoside. Conclusions The recombinant endoglucanase showed a threefold increase in extracellular enzyme activity compared with the wild-type strain. This result revealed the potential of endoglucanase expression in E. coli, which can be induced for the overexpression of the enzyme. The enzyme has a broad range of activity with high specificity toward cellulose.

The evolutionary strata of DARPP-32 tail implicates hierarchical functional expansion in higher vertebrates.

DARPP-32 (dopamine and adenosine 3', 5'-monophosphate-regulated phosphoprotein of 32 kDa), which belongs to PPP1R1 gene family, is known to act as an important integrator in dopamine-mediated neurotransmission via the inhibition of protein phosphatase-1 (PP1). Besides its neuronal roles, this protein also behaves as a key player in pathological and pharmacological aspects. Use of bioinformatics and phylogenetics approaches to further characterize the molecular features of DARPP-32 can guide future works. Predicted phosphorylation sites on DARPP-32 show conservation across vertebrates. Phylogenetics analysis indicates evolutionary strata of phosphorylation site acquisition at the C-terminus, suggesting functional expansion of DARPP-32, where more diverse signalling cues may involve in regulating DARPP-32 in inhibiting PP1 activity. Moreover, both phylogenetics and synteny analyses suggest de novo origination of PPP1R1 gene family via chromosomal rearrangement and exonization.