Expression, purification and virucidal activity of two recombinantisoforms of phospholipase A2from Crotalus durissus terrificus venom

AbstractThe global emergence and re-emergence of arthropod-borne viruses (arboviruses) over the past four decades have becomea public health crisis of international concern, especially in tropical and subtropical countries. A limited number of vaccinesagainst arboviruses are available for use in humans; therefore, there is an urgent need to develop antiviral compounds.Snake venoms are rich sources of bioactive compounds with potential for antiviral prospection. The major component ofCrotalus durissus terrificus venom is a heterodimeric complex called crotoxin, which is constituted by an inactive peptide(crotapotin) and a phospholipase A2(PLA2-CB). We showed previously the antiviral effect of PLA2-CB against denguevirus, yellow fever virus and other enveloped viruses. The aims of this study were to express two PLA2-CB isoforms in aprokaryotic system and to evaluate their virucidal effects. The sequences encoding the PLA2-CB isoforms were optimizedand cloned into a plasmid vector (pG21a) for recombinant protein expression. The recombinant proteins were expressedin the E. coli BL21(DE3) strain as insoluble inclusion bodies; therefore, the purification was performed under denaturingconditions, using urea for protein solubilization. The solubilized proteins were applied to a nickel affinity chromatographymatrix for binding. The immobilized recombinant proteins were subjected to an innovative protein refolding step, whichconsisted of the application of a decreasing linear gradient of urea and dithiothreitol (DTT) concentrations in combinationwith the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate hydrate (CHAPS) as a protein stabilizer.The refolded recombinant proteins showed phospholipase activity and virucidal effects against chikungunya virus, denguevirus, yellow fever virus and Zika virus

Interaction of the intrinsically disordered C?terminal domainof the sesbania mosaic virus RNA?dependent RNA polymerasewith the viral protein P10 in vitro: modulation of the oligomeric stateand polymerase activity

AbstractThe RNA-dependent RNA polymerase (RdRp) of sesbania mosaic virus (SeMV) was previously shown to interact withthe viral protein P10, which led to enhanced polymerase activity. In the present investigation, the equilibrium dissociationconstant for the interaction between the two proteins was determined to be 0.09 ?M using surface plasmon resonance, andthe disordered C-terminal domain of RdRp was shown to be essential for binding to P10. The association with P10 broughtabout a change in the oligomeric state of RdRp, resulting in reduced aggregation and increased polymerase activity. Interestingly,unlike the wild-type RdRp, C-terminal deletion mutants (C del 43 and C del 72) were found to exist predominantlyas monomers and were as active as the RdRp-P10 complex. Thus, either the deletion of the C-terminal disordered domainor its masking by binding to P10 results in the activation of polymerase activity. Further, deletion of the C-terminal 85 residuesof RdRp resulted in complete loss of activity. Mutation of a conserved tyrosine (RdRp Y480) within motif E, locatedbetween 72 and 85 residues from the C-terminus of RdRp, rendered the protein inactive, demonstrating the importance ofmotif E in RNA synthesis in vitro

Entrepreneurial ecosystem research: present debates and future directions

Abstract The purpose of this article is to review the emerging research on entrepreneurial ecosystem and to guide future research into this promising area. The study presents a critical review on the entrepreneurial ecosystem, starting from its very definition and antecedents. Combining prior research with building on the main concepts that constitute an entrepreneurial ecosystem, we have developed an original set of guidelines that can help scholars and practitioners seeking an answer to the following pressing question: BHow can we gain a comprehensive understanding of an entrepreneurial ecosystem?^. We will then discuss the opportunities for expanding our current knowledge on entrepreneurial ecosystems and describe the current debates and directions for future research. Lastly, we will provide guidelines that policymakers may take into consideration when designing and issuing support measures to promote entrepreneurship in their local ecosystems.Keywords Entrepreneurialecosystem.Entrepreneurship.Newventures.Syste